Cancer treatment with recombinant vector

ABSTRACT

This disclosure provides modified cytosine deaminases(CDs). The disclosure further relates to cells and vector expressing or comprising such modified CDs and methods of using such modified CDs in the treatment of disease and disorders. It further provides use of such modified CDs with a thymosin-alpha-1 polypeptide in the treatment of disease and disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/318,728, filed Mar. 29, 2010, the disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to replication competent retroviral vectors fortreating cell proliferative. The disclosure further relates to the useof such replication competent retroviral vectors and factors fordelivery and expression of heterologous nucleic acids.

BACKGROUND

Effective methods of delivering genes and heterologous nucleic acids tocells and subjects has been a goal researchers for scientificdevelopment and for possible treatments of diseases and disorders.

SUMMARY

The disclosure provides a therapeutic combination comprising athymosin-1-alpha polypeptide and a replication retroviral vector for usein the treatment of a subject comprising a cell proliferative disease ordisorder, wherein the replication competent retroviral vector comprisesa retroviral GAG protein; a retroviral POL protein; a retroviralenvelope; a retroviral polynucleotide comprising Long-Terminal Repeat(LTR) sequences at the 3′ end of the retroviral polynucleotide sequence,a promoter sequence at the 5′ end of the retroviral polynucleotide, saidpromoter being suitable for expression in a mammalian cell, a gagnucleic acid domain, a pol nucleic acid domain and an env nucleic aciddomain; a cassette comprising an internal ribosome entry site (IRES)operably linked to a heterologous polynucleotide, wherein the cassetteis positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domainencoding the retroviral envelope; and cis-acting sequences necessary forreverse transcription, packaging and integration in a target cell. Inone embodiment, the heterologous polynucleotide comprises a suicide genethat expresses a polypeptide that converts a non-toxic prodrug to atoxic drug. In another embodiment, the target cell is a cancer cell. Inyet another embodiment, the target cell comprises a cell proliferativedisorder. In a further embodiment, the cell proliferative disorder isselected from the group consisting of lung cancer, colon-rectum cancer,breast cancer, prostate cancer, urinary tract cancer, uterine cancer,brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomachcancer and ovarian cancer, rheumatoid arthritis or other autoimmunedisease. In one embodiment, the retroviral vector is administered priorto the thymosin-alpha-1 polypeptide. In another embodiment, theretroviral polynucleotide sequence is derived from murine leukemia virus(MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus(FeLV) Baboon endogenous retrovirus (BEV), porcine endogenous virus(PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus,Xenotropic murine leukemia virus-related virus (XMRV), avianreticuloendotheliosis virus (REV), or Gibbon ape leukemia virus (GALV).In yet another embodiment, the retroviral envelope is an amphotropic MLVenvelope. In one embodiment, the retrovirus is a gammaretrovirus. Inanother embodiment, the thymosin-alpha-1 polypeptide comprises at least85% identity to SEQ ID NO:73 and having a thymosin-alpha-1 activity. Inyet another embodiment, the heterologous polynucleotide encodes apolypeptide having cytosine deaminase activity. In one embodiment, theheterologous polynucleotide is selected from the group consisting of asuicide gene and an immunopotentiating gene. In any of the foregoingembodiments, the retrovirus further comprises an miRNA. In a specificembodiment, the replication competent retrovirus comprising a retroviralGAG protein; a retroviral POL protein; a retroviral envelope; aretroviral polynucleotide comprising Long-Terminal Repeat (LTR)sequences at the 3′ end of the retroviral polynucleotide sequence, apromoter sequence at the 5′ end of the retroviral polynucleotide, saidpromoter being suitable for expression in a mammalian cell, a gagnucleic acid domain, a pol nucleic acid domain and an env nucleic aciddomain; a cassette comprising an internal ribosome entry site (IRES)operably linked to a polynucleotide encoding cytosine deaminase, whereinthe cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleicacid domain encoding the retroviral envelope; and cis-acting sequencesnecessary for reverse transcription, packaging and integration in atarget cell. In any of the foregoing embodiments, the thymosin-1-alphaand retroviral vector are formulated for delivery simultaneously.

The disclosure also provides a method of treating a subject with a cellproliferative disorder comprising administering a thymosin-alpha-1polypeptide to the subject either before, during or after administrationof a replication competent retrovirus comprising a retroviral GAGprotein; a retroviral POL protein; a retroviral envelope; a retroviralpolynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′end of the retroviral polynucleotide sequence, a promoter sequence atthe 5′ end of the retroviral polynucleotide, said promoter beingsuitable for expression in a mammalian cell, a gag nucleic acid domain,a pol nucleic acid domain and an env nucleic acid domain; a cassettecomprising an internal ribosome entry site (IRES) operably linked to aheterologous polynucleotide, wherein the cassette is positioned 5′ tothe 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviralenvelope; and cis-acting sequences necessary for reverse transcription,packaging and integration in a target cell. In one embodiment, theheterologous polynucleotide comprises a suicide gene that expresses apolypeptide that converts a non-toxic prodrug to a toxic drug. Inanother embodiment, the target cell is a cancer cell. In yet anotherembodiment, the target cell comprises a cell proliferative disorder. Ina further embodiment, the cell proliferative disorder is selected fromthe group consisting of lung cancer, colon-rectum cancer, breast cancer,prostate cancer, urinary tract cancer, uterine cancer, brain cancer,head and neck cancer, pancreatic cancer, melanoma, stomach cancer andovarian cancer, rheumatoid arthritis or other autoimmune disease. In oneembodiment, the retroviral vector is administered prior to thethymosin-alpha-1 polypeptide. In another embodiment, the retroviralpolynucleotide sequence is derived from murine leukemia virus (MLV),Moloney murine leukemia virus (MoMLV), Feline leukemia virus (FeLV)Baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), thecat derived retrovirus RD114, squirrel monkey retrovirus, Xenotropicmurine leukemia virus-related virus (XMRV), avian reticuloendotheliosisvirus (REV), or Gibbon ape leukemia virus (GALV). In yet anotherembodiment, the retroviral envelope is an amphotropic MLV envelope. Inone embodiment, the retrovirus is a gammaretrovirus. In anotherembodiment, the thymosin-alpha-1 polypeptide comprises at least 85%identity to SEQ ID NO:73 and having a thymosin-alpha-1 activity. In yetanother embodiment, the heterologous polynucleotide encodes apolypeptide having cytosine deaminase activity. In one embodiment, theheterologous polynucleotide is selected from the group consisting of asuicide gene and an immunopotentiating gene. In any of the foregoingembodiments, the retrovirus further comprises an miRNA. In a specificembodiment, the replication competent retrovirus comprising a retroviralGAG protein; a retroviral POL protein; a retroviral envelope; aretroviral polynucleotide comprising Long-Terminal Repeat (LTR)sequences at the 3′ end of the retroviral polynucleotide sequence, apromoter sequence at the 5′ end of the retroviral polynucleotide, saidpromoter being suitable for expression in a mammalian cell, a gagnucleic acid domain, a pol nucleic acid domain and an env nucleic aciddomain; a cassette comprising an internal ribosome entry site (IRES)operably linked to a polynucleotide encoding cytosine deaminase, whereinthe cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleicacid domain encoding the retroviral envelope; and cis-acting sequencesnecessary for reverse transcription, packaging and integration in atarget cell. In any of the foregoing embodiments, the thymosin-1-alphaand retroviral vector are formulated for delivery simultaneously.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-C shows an alignment of the Wild-type yeast cytosine deaminase(SEQ ID NO: 2) and a cytosine deaminase of the disclosure (SEQ ID NO: 4)and other sequences of the disclosure (SEQ ID NOs:31-40).

FIG. 2 shows a graph of cell killing data showing that modified vectorsare more effective compared to the original wild type CD. The graph alsoshows that the new modified backbone (T5.0007) is more effective atkilling than the old backbone (pACE-CD). Also shown is a tablecataloguing the various vector constructs and their names

FIG. 3A-F shows (A) a schematic of a recombinant retroviral vector ofthe disclosure; (B) and (C) are plasmid maps of a polynucleotide of thedisclosure. (D) sequence of the vector encoding part of pAC3-yCD2 of thedisclosure. (E) schematics of recombinant vectors of the disclosure and(F) plasmid maps of vectors of the disclosure.

FIG. 4 shows that higher levels of yCD2 protein are observed compared towild type yCD protein in infected U-87 cells.

FIG. 5 shows that a vector of the disclosure is genetically stable after12 cycles of viral passages as assessed using PCR amplification. Thefigure also demonstrates that the vectors of the disclosure are morestable after longer passages compared to the vector pACE-CD (Kasahara etal.). In particular pAC3-CD is more stable than pACE-CD, demonstratingthat the changed backbone has made the vector more stable. In additionpACE-yCD1 (T5.0001) and -yCD2 (T5-0002) are very much more stable thanpAC-yCD, demonstrating that small and silent changes to the codingsequence of the transgene can have a very large effect on stability,leading to superior properties.

FIG. 6A-B shows cell killing assays and cytosine deaminase specificactivity of cells infected with different vectors. (A) shows thatcytosine deaminase and vector of the disclosure kill infected cells atleast as well and perhaps better than the original pACE-CD when U87infected cells are exposed to increasing levels of 5-FC. (B) shows thatthe specific CD activity of the disclosure (T5.0007, T5.0001 andT5.0002) are all increased compared to pACE-CD (T5.0000), and is in theorder T5.0000<T5.0007<T5.0001<T5.0002.

FIG. 7 shows U-87 tumors treated with CD vector of the disclosure invivo and explanted from mice treated with 4 cycles of 5-FC are stillsensitive to the drug.

FIG. 8 shows dosing information and therapeutic effect in a Kaplan-Meyersurvival analysis in a mouse model of brain cancer.

FIG. 9 shows dosing information and therapeutic effect in a Kaplan-Meyersurvival analysis in a syngeneic mouse model.

FIG. 10 shows a survival curve for combination therapy with thymosinalpha 1 and a replication competent retrovirus of the disclosureexpressing cytosine deaminase.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” includes aplurality of such cells and reference to “the agent” includes referenceto one or more agents known to those skilled in the art, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology Volume 152, (Academic Press, Inc., SanDiego, Calif.) (“Berger”); Sambrook et al., Molecular Cloning—ALaboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 1999) (“Ausubel”). Examples ofprotocols sufficient to direct persons of skill through in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification and otherRNA polymerase mediated techniques (e.g., NASBA), e.g., for theproduction of the homologous nucleic acids of the disclosure are foundin Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987)U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: AGuide to Methods and Applications (Academic Press Inc. San Diego,Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci.USA 87: 1874; Lomell et al. (1989) J. Clin. Chem 35: 1826; Landegren etal. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene89:117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564.Improved methods for cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Improved methodsfor amplifying large nucleic acids by PCR are summarized in Cheng et al.(1994) Nature 369: 684-685 and the references cited therein, in whichPCR amplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook andBerger, all supra.

The publications discussed throughout the text are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior disclosure.

The disclosure provides methods and compositions useful for treatingcell proliferative diseases and disorders. The disclosure providesreplication competent retroviral vectors for gene delivery andcombination therapies.

The terms “vector”, “vector construct” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors typically comprise the DNA of a transmissible agent,into which foreign DNA encoding a protein is inserted by restrictionenzyme technology. A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA that canreadily accept additional (foreign) DNA and which can readily introducedinto a suitable host cell. A large number of vectors, including plasmidand fungal vectors, have been described for replication and/orexpression in a variety of eukaryotic and prokaryotic hosts.Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids,pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids(Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs,Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g., antibiotic resistance, and one or moreexpression cassettes.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Apolynucleotide or polypeptide is expressed recombinantly, for example,when it is expressed or produced in a foreign host cell under thecontrol of a foreign or native promoter, or in a native host cell underthe control of a foreign promoter.

The disclosure provides replication competent viral vectors that containa heterologous polynucleotide encoding, for example, a cytosinedeaminase or mutant thereof that can be delivered to a cell or subject.The viral vector can be an adenoviral vector, a measles vector, a herpesvector, a retroviral vector (including a lentiviral vector), arhabdoviral vector such as a Vesicular Stomatitis viral vector, areovirus vector, a Seneca Valley Virus vector, a poxvirus vector(including animal pox or vaccinia derived vectors), a parvovirus vector(including an AAV vector), an alphavirus vector or other viral vectorknown to one skilled in the art (see also, e.g., Concepts in GeneticMedicine, ed. Boro Dropulic and Barrie Carter, Wiley, 2008, Hoboken,N.J.; The Development of Human Gene Therapy, ed. Theodore Friedmann,Cold Springs Harbor Laboratory Press, Cold springs Harbor, N.Y., 1999;Gene and Cell Therapy, ed. Nancy Smyth Templeton, Marcel Dekker Inc.,New York, N.Y., 2000 and Gene Therapy: Therapeutic Mechanism andStrategies, ed. Nancy Smyth Templetone and Danilo D Lasic, MarcelDekker, Inc., New York, N.Y., 2000; the disclosures of which areincorporated herein by reference).

In one embodiment, the viral vector can be a replication competentretroviral vector capable of infecting only replicating mammalian cells.In one embodiment, a replication competent retroviral vector comprisesan internal ribosomal entry site (IRES) 5′ to the heterologouspolynucleotide encoding, e.g., a cytosine deaminase or the like. In oneembodiment, the polynucleotide is 3′ to a ENV polynucleotide of aretroviral vector. In one embodiment the viral vector is a retroviralvector capable of infecting target cells multiple times (5 or more perdiploid cell).

The disclosure also provides replication competent retroviral vectorshaving increased stability relative to prior retroviral vectors. Suchincreased stability during infection and replication is important forthe treatment of cell proliferative disorders. The combination oftransduction efficiency, transgene stability and target selectivity isprovided by the replication competent retrovirus. The compositions andmethods provide insert stability and maintain transcription activity ofthe transgene and the translational viability of the encodedpolypeptide.

The disclosure provides modified retroviral vectors. The modifiedretroviral vectors can be derived from members of the retroviridaefamily. The Retroviridae family consists of three groups: thespumaviruses-(or foamy viruses) such as the human foamy virus (HFV); thelentiviruses, as well as visna virus of sheep; and the oncoviruses(although not all viruses within this group are oncogenic). The term“lentivirus” is used in its conventional sense to describe a genus ofviruses containing reverse transcriptase. The lentiviruses include the“immunodeficiency viruses” which include human immunodeficiency virus(HIV) type 1 and type 2 (HIV-1 and HIV-2) and simian immunodeficiencyvirus (SIV). The oncoviruses have historically been further subdividedinto groups A, B, C and D on the basis of particle morphology, as seenunder the electron microscope during viral maturation. A-type particlesrepresent the immature particles of the B- and D-type viruses seen inthe cytoplasm of infected cells. These particles are not infectious.B-type particles bud as mature virion from the plasma membrane by theenveloping of intracytoplasmic A-type particles. At the membrane theypossess a toroidal core of 75 nm, from which long glycoprotein spikesproject. After budding, B-type particles contain an eccentricallylocated, electron-dense core. The prototype B-type virus is mousemammary tumor virus (MMTV). No intracytoplasmic particles can beobserved in cells infected by C-type viruses. Instead, mature particlesbud directly from the cell surface via a crescent ‘C’-shapedcondensation which then closes on itself and is enclosed by the plasmamembrane. Envelope glycoprotein spikes may be visible, along with auniformly electron-dense core. Budding may occur from the surface plasmamembrane or directly into intracellular vacuoles. The C-type viruses arethe most commonly studied and include many of the avian and murineleukemia viruses (MLV). Bovine leukemia virus (BLV), and the humanT-cell leukemia viruses types I and II (HTLV-I/II) are similarlyclassified as C-type particles because of the morphology of theirbudding from the cell surface. However, they also have a regularhexagonal morphology and more complex genome structures than theprototypic C-type viruses such as the murine leukemia viruses (MLV).D-type particles resemble B-type particles in that they show asring-like structures in the infected cell cytoplasm, which bud from thecell surface, but the virion incorporate short surface glycoproteinspikes. The electron-dense cores are also eccentrically located withinthe particles. Mason Pfizer monkey virus (MPMV) is the prototype D-typevirus.

Retroviruses have been classified in various ways but the nomenclaturehas been standardized in the last decade (see ICTVdB—The Universal VirusDatabase, v 4 on the World Wide Web (www) atncbi.nlm.nih.gov/ICTVdb/ICTVdB/ and the text book “Retroviruses” EdsCoffin, Hughs and Varmus, Cold Spring Harbor Press 1997; the disclosuresof which are incorporated herein by reference). In one embodiment, thereplication competent retroviral vector can comprise an Orthoretrovirusor more typically a gamma retrovirus vector.

Retroviruses are defined by the way in which they replicate theirgenetic material. During replication the RNA is converted into DNA.Following infection of the cell a double-stranded molecule of DNA isgenerated from the two molecules of RNA which are carried in the viralparticle by the molecular process known as reverse transcription. TheDNA form becomes covalently integrated in the host cell genome as aprovirus, from which viral RNAs are expressed with the aid of cellularand/or viral factors. The expressed viral RNAs are packaged intoparticles and released as infectious virion.

The retrovirus particle is composed of two identical RNA molecules. Eachwild-type genome has a positive sense, single-stranded RNA molecule,which is capped at the 5′ end and polyadenylated at the 3′ tail. Thediploid virus particle contains the two RNA strands complexed with gagproteins, viral enzymes (pol gene products) and host tRNA moleculeswithin a ‘core’ structure of gag proteins. Surrounding and protectingthis capsid is a lipid bilayer, derived from host cell membranes andcontaining viral envelope (env) proteins. The env proteins bind to acellular receptor for the virus and the particle typically enters thehost cell via receptor-mediated endocytosis and/or membrane fusion.

After the outer envelope is shed, the viral RNA is copied into DNA byreverse transcription. This is catalyzed by the reverse transcriptaseenzyme encoded by the pol region and uses the host cell tRNA packagedinto the virion as a primer for DNA synthesis. In this way the RNAgenome is converted into the more complex DNA genome.

The double-stranded linear DNA produced by reverse transcription may, ormay not, have to be circularized in the nucleus. The provirus now hastwo identical repeats at either end, known as the long terminal repeats(LTR). The termini of the two LTR sequences produces the site recognizedby a pol product—the integrase protein—which catalyzes integration, suchthat the provirus is always joined to host DNA two base pairs (bp) fromthe ends of the LTRs. A duplication of cellular sequences is seen at theends of both LTRs, reminiscent of the integration pattern oftransposable genetic elements. Retroviruses can integrate their DNAs atmany sites in host DNA, but different retroviruses have differentintegration site preferences. HIV-1 and simian immunodeficiency virusDNAs preferentially integrate into expressed genes, murine leukemiavirus (MLV) DNA preferentially integrates near transcriptional startsites (TSSs), and avian sarcoma leukosis virus (ASLV) and human T cellleukemia virus (HTLV) DNAs integrate nearly randomly, showing a slightpreference for genes (Derse D, et al. (2007) Human T-cell leukemia virustype 1 integration target sites in the human genome: comparison withthose of other retroviruses. J Virol 81:6731-6741; Lewinski M K, et al.(2006) Retroviral DNA integration: viral and cellular determinants oftarget-site selection. PLoS Pathog 2:e601).

Transcription, RNA splicing and translation of the integrated viral DNAis mediated by host cell proteins. Variously spliced transcripts aregenerated. In the case of the human retroviruses HIV-1/2 and HTLV-I/IIviral proteins are also used to regulate gene expression. The interplaybetween cellular and viral factors is a factor in the control of viruslatency and the temporal sequence in which viral genes are expressed.

Retroviruses can be transmitted horizontally and vertically. Efficientinfectious transmission of retroviruses requires the expression on thetarget cell of receptors which specifically recognize the viral envelopeproteins, although viruses may use receptor-independent, nonspecificroutes of entry at low efficiency. Normally a viral infection leads to asingle or few copies of viral genome per cell because of receptormasking or down-regulation that in turn leads to resistance tosuperinfection (Ch3 p 104 in “Retroviruses” J M Coffin, S H Hughes, & HE Varmus 1997 Cold Spring Harbor Laboratory Press, Cold Spring HarborN.Y.; Fan et al. J. Virol 28:802, 1978). By manipulating the situationin tissue culture it is possible to get some level of multiple infectionbut this is typically less than 5 copies/diploid genome. In addition,the target cell type must be able to support all stages of thereplication cycle after virus has bound and penetrated. Verticaltransmission occurs when the viral genome becomes integrated in the germline of the host. The provirus will then be passed from generation togeneration as though it were a cellular gene. Hence endogenousproviruses become established which frequently lie latent, but which canbecome activated when the host is exposed to appropriate agents.

In many situations for using a recombinant replication competentretrovirus therapeutically, it is advantageous to have high levels ofexpression of the transgene that is encoded by the recombinantreplication competent retrovirus. For example, with a prodrug activatinggene such as the cytosine deaminase gene it is advantageous to havehigher levels of expression of the CD protein in a cell so that theconversion of the prodrug 5-FC to 5-FU is more efficient. The disclosureprovides recombinant replication competent retroviruses capable ofinfecting a target cell or target cell population multiple timesresulting in an average number of copies/diploid genome of 5 or greater.Also provided are methods of treating a cell proliferative disorder,using a recombinant replication competent retrovirus capable ofinfecting a target cell or target cell population multiple timesresulting in an average number of copies/diploid genome of 5 or greater.In further embodiments, a combination therapy comprisingthymosin-alpha-1 is used to promote apoptosis and therapeutic effects ofa RCR of the disclosure.

As mentioned above, the integrated DNA intermediate is referred to as aprovirus. Prior gene therapy or gene delivery systems use methods andretroviruses that require transcription of the provirus and assemblyinto infectious virus while in the presence of an appropriate helpervirus or in a cell line containing appropriate sequences enablingencapsidation without coincident production of a contaminating helpervirus. As described below, a helper virus is not required for theproduction of the recombinant retrovirus of the disclosure, since thesequences for encapsidation are provided in the genome thus providing areplication competent retroviral vector for gene delivery or therapy.

Other existing replication competent retroviral vectors also tend to beunstable and lose sequences during horizontal or vertical transmissionto an infected cell or host cell and during replication. This may be duein-part from the presence of extra nucleotide sequences that includerepeats or which reduce the efficiency of a polymerase.

The retroviral genome and the proviral DNA of the disclosure have atleast three genes: the gag, the pol, and the env, these genes may beflanked by one or two long terminal (LTR) repeat, or in the provirus areflanked by two long terminal repeat (LTR) and sequences containingcis-acting sequences such as psi. The gag gene encodes the internalstructural (matrix, capsid, and nucleocapsid) proteins; the pol geneencodes the RNA-directed DNA polymerase (reverse transcriptase),protease and integrase; and the env gene encodes viral envelopeglycoproteins. The 5′ and/or 3′ LTRs serve to promote transcription andpolyadenylation of the virion RNAs. The LTR contains all othercis-acting sequences necessary for viral replication. Lentiviruses haveadditional genes including vif, vpr, tat, rev, vpu, nef, and vpx (inHIV-1, HIV-2 and/or SIV).

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virion) are missing from the viral genome, the result isa cis defect which prevents encapsidation of genomic viral RNA. Thistype of modified vector is what has typically been used in prior genedelivery systems (i.e., systems lacking elements which are required forencapsidation of the virion) as ‘helper’ elements providing viralproteins in trans that package a non-replicating, but packageable, RNAgenome.

In a first embodiment, the disclosure provides a recombinant retroviruscapable of infecting a dividing cell or a cell having a cellproliferative disorder. The recombinant replication competent retrovirusof the disclosure comprises a polynucleotide sequence encoding a viralGAG, a viral POL, a viral ENV, a heterologous polynucleotide preceded byan internal ribosome entry site (IRES) encapsulated within a virion. Inone embodiment the heterologous polynucleotide encodes a polypeptidehaving cytosine deaminase activity. In yet another embodiment, apolypeptide having thymosin-alpha-1 activity is administeredsimultaneously, prior to, or after administration of the retroviralvector.

The phrase “non-dividing” cell refers to a cell that does not go throughmitosis. Non-dividing cells may be blocked at any point in the cellcycle, (e.g., G₀/G₁, G_(1/S), G_(2/M)), as long as the cell is notactively dividing. For ex vivo infection, a dividing cell can be treatedto block cell division by standard techniques used by those of skill inthe art, including, irradiation, aphidocolin treatment, serumstarvation, and contact inhibition. However, it should be understoodthat ex vivo infection is often performed without blocking the cellssince many cells are already arrested (e.g., stem cells). For example, arecombinant lentivirus vector is capable of infecting non-dividingcells. Examples of pre-existing non-dividing cells in the body includeneuronal, muscle, liver, skin, heart, lung, and bone marrow cells, andtheir derivatives. For dividing cells onco-retroviral vectors can beused.

By “dividing” cell is meant a cell that undergoes active mitosis, ormeiosis. Such dividing cells include stem cells, skin cells (e.g.,fibroblasts and keratinocytes), gametes, and other dividing cells knownin the art. Of particular interest and encompassed by the term dividingcell are cells having cell proliferative disorders, such as neoplasticcells. The term “cell proliferative disorder” refers to a conditioncharacterized by an abnormal number of cells. The condition can includeboth hypertrophic (the continual multiplication of cells resulting in anovergrowth of a cell population within a tissue) and hypotrophic (a lackor deficiency of cells within a tissue) cell growth or an excessiveinflux or migration of cells into an area of a body. The cellpopulations are not necessarily transformed, tumorigenic or malignantcells, but can include normal cells as well. Cell proliferativedisorders include disorders associated with an overgrowth of connectivetissues, such as various fibrotic conditions, including scleroderma,arthritis and liver cirrhosis. Cell proliferative disorders includeneoplastic disorders such as head and neck carcinomas. Head and neckcarcinomas would include, for example, carcinoma of the mouth,esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands,nose, paranasal sinuses, nasopharynx, superior nasal vault and sinustumors, esthesioneuroblastoma, squamous call cancer, malignant melanoma,sinonasal undifferentiated carcinoma (SNUC), brain (includingglioblastomas) or blood neoplasia. Also included are carcinoma's of theregional lymph nodes including cervical lymph nodes, prelaryngeal lymphnodes, pulmonary juxtaesophageal lymph nodes and submandibular lymphnodes (Harrison's Principles of Internal Medicine (eds., Isselbacher, etal., McGraw-Hill, Inc., 13th Edition, pp 1850-1853, 1994). Other cancertypes, include, but are not limited to, lung cancer, colon-rectumcancer, breast cancer, prostate cancer, urinary tract cancer, uterinecancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma,stomach cancer, skin cancer and ovarian cancer. The cell proliferativedisease also includes rheumatoid arthritis (O'Dell NEJM 350:2591 2004)and other auto-immune disorders (Mackay et al NEJM 345:340 2001) thatare often characterized by inappropriate proliferation of cells of theimmune system.

The heterologous nucleic acid sequence is operably linked to an IRES. Asused herein, the term “heterologous” nucleic acid sequence or transgenerefers to (i) a sequence that does not normally exist in a wild-typeretrovirus, (ii) a sequence that originates from a foreign species, or(iii) if from the same species, it may be substantially modified fromits original form. Alternatively, an unchanged nucleic acid sequencethat is not normally expressed in a cell is a heterologous nucleic acidsequence.

Depending upon the intended use of the retroviral vector of thedisclosure any number of heterologous polynucleotide or nucleic acidsequences may be inserted into the retroviral vector. For example, forin vitro studies commonly used marker genes or reporter genes may beused, including, antibiotic resistance and fluorescent molecules (e.g.,GFP). Additional polynucleotide sequences encoding any desiredpolypeptide sequence may also be inserted into the vector of thedisclosure. Where in vivo delivery of a heterologous nucleic acidsequence is sought both therapeutic and non-therapeutic sequences may beused. For example, the heterologous sequence can encode a therapeuticmolecule including antisense molecules (miRNA, siRNA) or ribozymesdirected to a particular gene associated with a cell proliferativedisorder or other gene-associated disease or disorder, the heterologoussequence can be a suicide gene (e.g., HSV-tk or PNP or cytosinedeaminase; either modified or unmodified), a growth factor or atherapeutic protein (e.g., Factor IX, IL2, and the like). Othertherapeutic proteins applicable to the disclosure are easily identifiedin the art.

In one embodiment, the heterologous polynucleotide within the vectorcomprises a cytosine deaminase that has been optimized for expression ina human cell. In a further embodiment, the cytosine deaminase comprisesa sequence that has been human codon optimized and comprises mutationsthat increase the cytosine deaminase's stability (e.g., reduceddegradation or increased thermo-stability) compared to a wild-typecytosine deaminase. In yet another embodiment, the heterologouspolynucleotide encodes a fusion construct comprising a cytosinedeaminase (either human codon optimized or non-optimized, either mutatedor non-mutated) operably linked to a polynucleotide encoding apolypeptide having UPRT or OPRT activity. In another embodiment, theheterologous polynucleotide comprises a CD polynucleotide of thedisclosure (e.g., SEQ ID NO:3, 5, 11, 13, 15, or 17).

In another embodiment, replication competent retroviral vector cancomprise a heterologous polynucleotide encoding a polypeptide comprisinga cytosine deaminase (as described herein) and may further comprise apolynucleotide comprising a miRNA or siRNA molecule either as part ofthe primary transcript from the viral promoter or linked to a promoter,which can be cell-type or tissue specific.

For examples, miRNAs that are down-regulated in cancers could be usefulas anticancer agents. Examples include mir-128-1, let-7, miR-26,miR-124, and miR-137 (Esquela-Kerscher et al., 2008 Cell Cycle 7,759-764; Kumar et al., 2008 Proc Natl Acad Sci USA 105, 3903-3908; Kotaet al., 2009 Cell 137, 1005-1017; Silber et al., 2008 BMC Medicine 6:141-17). miR-128 expression has reported to be enriched in the centralnervous system and has been observed to be down-regulated inglioblastomas (Sempere et al., 2004 Genome Biology 5:R13.5-11; Godlewskiet al., 2008 Cancer Res 68: (22) 9125-9130). miR-128 is encoded by twodistinct genes, miR-128-1 and miR-128-2. Both are processed intoidentical mature sequence. Bmi-1 and E2F3a have been reported to be thedirect targets of miR-128 (Godlewski et al., 2008 Cancer Res 68: (22)9125-9130; Zhang et al., 2009 J. Mol Med 87:43-51). In addition, Bmi-1expression has been observed to be up-regulated in a variety of humancancers, including gliomas, mantle cell lymphomas, non-small cell lungcancer B-cell non-Hodgkin's lymphoma, breast, colorectal and prostatecancer. Furthermore, Bmi-1 has been demonstrated to be required for theself-renewal of stem cells from diverse tissues, including neuronal stemcells as well as “stem-like” cell population in gliomas.

In one embodiment, the disclosure provides a recombinant replicationcompetent retroviral vector that contains a single copy of themiR-142-3p target sequence (142-3pT, SEQ ID NO:35) downstream of thetransgene, such as yCD2 or GFP, linked to the IRES. In addition tomiR181 and miR-223, the target sequence of other tissue or cell-enrichedmiRNA can be incorporated into the vector to restrict viral spread inspecific tissue or cell type manner. For example, miR-133 and miR206expressions are highly enriched in muscle cells (Kelly et al., 2008Nature Medicine 14:11 1278-1283.

In another embodiment, the disclosure provides a recombinant replicationcompetent retroviral vector that contains 4 copies of the 142-3pT (SEQID NO: 36) downstream of the transgene, such as yCD2 or GFP, linked tothe IRES. In addition to miR181 and miR-223, the target sequence ofother tissue or cell-enriched miRNA can be incorporated into the vectorto restrict viral spread in specific tissue or cell type manner.

In yet further embodiments, the heterologous polynucleotide may comprisea cytokine such as an interleukin, interferon gamma or the like.Cytokines that may expressed from a retroviral vector of the disclosureinclude, but are not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21, anti-CD40, CD40L,IFN-gamma and TNF-alpha, soluble forms of TNF-alpha, lymphotoxin-alpha(LT-alpha, also known as TNF-beta), LT-beta (found in complexheterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL,DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328),AIM-I (International Publication No. WO 97/33899), endokine-alpha(International Publication No. WO 98/07880), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9(International Publication No. WO 98/56892), TR10 (InternationalPublication No. WO 98/54202), 312C2 (International Publication No. WO98/06842), and TR12, and soluble forms CD154, CD70, and CD153.Angiogenic proteins may be useful in some embodiments, particularly forprotein production from cell lines. Such angiogenic factors include, butare not limited to, Glioma Derived Growth Factor (GDGF), PlateletDerived Growth Factor-A (PDGF-A), Platelet Derived Growth Factor-B(PDGF-B), Placental Growth Factor (PIGF), Placental Growth Factor-2(PIGF-2), Vascular Endothelial Growth Factor (VEGF), VascularEndothelial Growth Factor-A (VEGF-A), Vascular Endothelial GrowthFactor-2 (VEGF-2), Vascular Endothelial Growth Factor B (VEGF-3),Vascular Endothelial Growth Factor B-1 86 (VEGF-B186), VascularEndothelial Growth Factor-D (VEGF-D), Vascular Endothelial GrowthFactor-D (VEGF-D), and Vascular Endothelial Growth Factor-E (VEGF-E).Fibroblast Growth Factors may be delivered by a vector of the disclosureand include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5,FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, andFGF-15. Hematopoietic growth factors may be delivered using vectors ofthe disclosure, such growth factors include, but are not limited to,granulocyte macrophage colony stimulating factor (GM-CSF)(sargramostim), granulocyte colony stimulating factor (G-CSF)(filgrastim), macrophage colony stimulating factor (M-CSF, CSF-1)erythropoietin (epoetin alfa), stem cell factor (SCF, c-kit ligand,steel factor), megakaryocyte colony stimulating factor, PIXY321 (aGMCSF/IL-3) fusion protein and the like.

The term “regulatory nucleic acid sequence” refers collectively topromoter sequences, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, origins of replication,enhancers and the like, which collectively provide for the replication,transcription and translation of a coding sequence in a recipient cell.Not all of these control sequences need always be present so long as theselected coding sequence is capable of being replicated, transcribed andtranslated in an appropriate host cell. One skilled in the art canreadily identify regulatory nucleic acid sequence from public databasesand materials. Furthermore, one skilled in the art can identify aregulatory sequence that is applicable for the intended use, forexample, in vivo, ex vivo, or in vitro.

An internal ribosome entry sites (“IRES”) refers to a segment of nucleicacid that promotes the entry or retention of a ribosome duringtranslation of a coding sequence usually 3′ to the IRES. In someembodiments the IRES may comprise a splice acceptor/donor site, however,preferred IRESs lack a splice acceptor/donor site. Normally, the entryof ribosomes into messenger RNA takes place via the cap located at the5′ end of all eukaryotic mRNAs. However, there are exceptions to thisuniversal rule. The absence of a cap in some viral mRNAs suggests theexistence of alternative structures permitting the entry of ribosomes atan internal site of these RNAs. To date, a number of these structures,designated IRES on account of their function, have been identified inthe 5′ noncoding region of uncapped viral mRNAs, such as that, inparticular, of picornaviruses such as the poliomyelitis virus (Pelletieret al., 1988, Mol. Cell. Biol., 8, 1103-1112) and the EMCV virus(encephalo-myocarditis virus (Jang et al., J. Virol., 1988, 62,2636-2643). The disclosure provides the use of an IRES in the context ofa replication-competent retroviral vector.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. The regulatory sequence may behomologous or heterologous to the desired gene sequence. For example, awide range of promoters may be utilized, including viral or mammalianpromoter as described above.

The heterologous nucleic acid sequence is typically under control ofeither the viral LTR promoter-enhancer signals or an internal promoter,and retained signals within the retroviral LTR can still bring aboutefficient integration of the vector into the host cell genome.Accordingly, the recombinant retroviral vectors of the disclosure, thedesired sequences, genes and/or gene fragments can be inserted atseveral sites and under different regulatory sequences. For example, asite for insertion can be the viral enhancer/promoter proximal site(i.e., 5′ LTR-driven gene locus). Alternatively, the desired sequencescan be inserted into a regulatory sequence distal site (e.g., the IRESsequence 3′ to the env gene) or where two or more heterologous sequencesare present one heterologous sequence may be under the control of afirst regulatory region and a second heterologous sequence under thecontrol of a second regulatory region. Other distal sites include viralpromoter sequences, where the expression of the desired sequence orsequences is through splicing of the promoter proximal cistron, aninternal heterologous promoter as SV40 or CMV, or an internal ribosomeentry site (IRES) can be used.

In one embodiment, the retroviral genome of the disclosure contains anIRES comprising a cloning site downstream of the IRES for insertion of adesired/heterologous polynucleotide. In one embodiment, the IRES islocated 3′ to the env gene in the retroviral vector, but 5′ to thedesired heterologous polynucleotide. Accordingly, a heterologouspolynucleotide encoding a desired polypeptide may be operably linked tothe IRES.

In another embodiment, a targeting polynucleotide sequence is includedas part of the recombinant retroviral vector of the disclosure. Thetargeting polynucleotide sequence is a targeting ligand (e.g., peptidehormones such as heregulin, a single-chain antibodies, a receptor or aligand for a receptor), a tissue-specific or cell-type specificregulatory element (e.g., a tissue-specific or cell-type specificpromoter or enhancer), or a combination of a targeting ligand and atissue-specific/cell-type specific regulatory element. Preferably, thetargeting ligand is operably linked to the env protein of theretrovirus, creating a chimeric retroviral env protein. The viral GAG,viral POL and viral ENV proteins can be derived from any suitableretrovirus (e.g., MLV or lentivirus-derived). In another embodiment, theviral ENV protein is non-retrovirus-derived (e.g., CMV or VSV).

In one embodiment, the recombinant retrovirus of the disclosure isgenetically modified in such a way that the virus is targeted to aparticular cell type (e.g., smooth muscle cells, hepatic cells, renalcells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrowcells, chondrocyte, epithelial cells, intestinal cells, mammary cells,neoplastic cells, glioma cells, neuronal cells and others known in theart) such that the recombinant genome of the retroviral vector isdelivered to a target non-dividing, a target dividing cell, or a targetcell having a cell proliferative disorder.

In one embodiment, the retroviral vector is targeted to the cell bybinding to cells having a molecule on the external surface of the cell.This method of targeting the retrovirus utilizes expression of atargeting ligand on the coat of the retrovirus to assist in targetingthe virus to cells or tissues that have a receptor or binding moleculewhich interacts with the targeting ligand on the surface of theretrovirus. After infection of a cell by the virus, the virus injectsits nucleic acid into the cell and the retrovirus genetic material canintegrate into the host cell genome.

In another embodiment, targeting uses cell- or tissue-specificregulatory elements to promote expression and transcription of the viralgenome in a targeted cell which actively utilizes the regulatoryelements, as described more fully below. The transferred retrovirusgenetic material is then transcribed and translated into proteins withinthe host cell. The targeting regulatory element is typically linked tothe 5′ and/or 3′ LTR, creating a chimeric LTR.

By inserting a heterologous polynucleotide of interest into the viralvector of the disclosure, along with another gene which encodes, forexample, the ligand for a receptor on a specific target cell, the vectoris now target specific. Viral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Targetingcan be accomplished by using an antibody to target the viral vector.Those of skill in the art will know of, or can readily ascertain,specific polynucleotide sequences which can be inserted into the viralgenome or proteins which can be attached to a viral envelope to allowtarget specific delivery of the viral vector containing the nucleic acidsequence of interest.

Thus, the disclosure includes in one embodiment, a chimeric env proteincomprising a retroviral ENV protein operably linked to a targetingpolypeptide. The targeting polypeptide can be a cell specific receptormolecule, a ligand for a cell specific receptor, an antibody or antibodyfragment to a cell specific antigenic epitope or any other ligand easilyidentified in the art which is capable of binding or interacting with atarget cell. Examples of targeting polypeptides or molecules includebivalent antibodies using biotin-streptavidin as linkers (Etienne-Julanet al., J. Of General Virol., 73, 3251-3255 (1992); Roux et al., Proc.Natl. Acad. Sci USA 86, 9079-9083 (1989)), recombinant virus containingin its envelope a sequence encoding a single-chain antibody variableregion against a hapten (Russell et al., Nucleic Acids Research, 21,1081-1085 (1993)), cloning of peptide hormone ligands into theretrovirus envelope (Kasahara et al., Science, 266, 1373-1376 (1994)),chimeric EPO/env constructs (Kasahara et al., 1994), single-chainantibody against the low density lipoprotein (LDL) receptor in theecotropic MLV envelope, resulting in specific infection of HeLa cellsexpressing LDL receptor (Somia et al., Proc. Natl. Acad. Sci USA, 92,7570-7574 (1995)), similarly the host range of ALV can be altered byincorporation of an integrin ligand, enabling the virus to now crossspecies to specifically infect rat glioblastoma cells (Valsesia-Wittmannet al., J. Virol. 68, 4609-4619 (1994)), and Dornberg and co-workers(Chu and Dornburg, J. Virol 69, 2659-2663 (1995); M. Engelstadter et al.Gene Therapy 8, 1202-1206 (2001)) have reported tissue-specifictargeting of spleen necrosis virus (SNV), an avian retrovirus, usingenvelopes containing single-chain antibodies directed against tumormarkers.

The disclosure provides a method of producing a recombinant retroviruscapable of infecting a target cell comprising transfecting a suitablehost cell with the following: a vector comprising a polynucleotidesequence encoding a viral gag, a viral pol and a viral env, and aheterologous polynucleotide, operably linked to a regulatory nucleicacid sequence, and recovering the recombinant virus.

The retrovirus and methods of the disclosure provide a replicationcompetent retrovirus that does not require helper virus or additionalnucleic acid sequence or proteins in order to propagate and producevirion. For example, the nucleic acid sequences of the retrovirus of thedisclosure encode a group specific antigen and reverse transcriptase,(and integrase and protease-enzymes necessary for maturation and reversetranscription), respectively, as discussed above. The viral gag and polcan be derived from a lentivirus, such as HIV or an oncovirus orgammaretrovirus such as MoMLV. In addition, the nucleic acid genome ofthe retrovirus of the disclosure includes a sequence encoding a viralenvelope (ENV) protein. The env gene can be derived from anyretroviruses. The env may be an amphotropic envelope protein whichallows transduction of cells of human and other species, or may be anecotropic envelope protein, which is able to transduce only mouse andrat cells. Further, it may be desirable to target the recombinant virusby linkage of the envelope protein with an antibody or a particularligand for targeting to a receptor of a particular cell-type. Asmentioned above, retroviral vectors can be made target specific byinserting, for example, a glycolipid, or a protein. Targeting is oftenaccomplished by using an antibody to target the retroviral vector to anantigen on a particular cell-type (e.g., a cell type found in a certaintissue, or a cancer cell type). Those of skill in the art will know of,or can readily ascertain without undue experimentation, specific methodsto achieve delivery of a retroviral vector to a specific target. In oneembodiment, the env gene is derived from a non-retrovirus (e.g., CMV orVSV). Examples of retroviral-derived env genes include, but are notlimited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon apeleukemia virus (GaLV), human immunodeficiency virus (HIV) and RousSarcoma Virus (RSV). Other env genes such as Vesicular stomatitis virus(VSV) (Protein G), cytomegalovirus envelope (CMV), or influenza virushemagglutinin (HA) can also be used.

In one embodiment, the retroviral genome is derived from anonco-retrovirus, and more particularly a mammalian onco-retrovirus. In afurther embodiment, the retroviral genome is derived from a gammaretrovirus, and more particularly a mammalian gamma retrovirus. By“derived” is meant that the parent polynucleotide sequence is anwild-type oncovirus which has been modified by insertion or removal ofnaturally occurring sequences (e.g., insertion of an IRES, insertion ofa heterologous polynucleotide encoding a polypeptide or inhibitorynucleic acid of interest, swapping of a more effective promoter from adifferent retrovirus or virus in place of the wild-type promoter and thelike).

In another embodiment, the disclosure provides retroviral vectors thatare targeted using regulatory sequences. Cell- or tissue-specificregulatory sequences (e.g., promoters) can be utilized to targetexpression of gene sequences in specific cell populations. Suitablemammalian and viral promoters for the disclosure are described elsewhereherein. Accordingly, in one embodiment, the disclosure provides aretrovirus having tissue-specific promoter elements at the 5′ end of theretroviral genome. Typically, the tissue-specific regulatoryelements/sequences are in the U3 region of the LTR of the retroviralgenome, including for example cell- or tissue-specific promoters andenhancers to neoplastic cells (e.g., tumor cell-specific enhancers andpromoters), and inducible promoters (e.g., tetracycline).

Transcription control sequences of the disclosure can also includenaturally occurring transcription control sequences naturally associatedwith a gene encoding a superantigen, a cytokine or a chemokine.

In some circumstances, it may be desirable to regulate expression. Forexample, different viral promoters with varying strengths of activitymay be utilized depending on the level of expression desired. Inmammalian cells, the CMV immediate early promoter if often used toprovide strong transcriptional activation. Modified versions of the CMVpromoter that are less potent have also been used when reduced levels ofexpression of the transgene are desired. When expression of a transgenein hematopoietic cells is desired, retroviral promoters such as the LTRsfrom MLV or MMTV can be used. Other viral promoters that can be usedinclude SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such asfrom the E1A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus,HSV-TK, and avian sarcoma virus.

Similarly tissue specific or selective promoters may be used to effecttranscription in specific tissues or cells so as to reduce potentialtoxicity or undesirable effects to non-targeted tissues. For example,promoters such as the PSA, probasin, prostatic acid phosphatase orprostate-specific glandular kallikrein (hK2) may be used to target geneexpression in the prostate. The Whey accessory protein (WAP) may be usedfor breast tissue expression (Andres et al., PNAS 84:1299-1303, 1987).Other promoters/regulatory domains that can be used are set forth inTable 1.

“Tissue-specific regulatory elements” are regulatory elements (e.g.,promoters) that are capable of driving transcription of a gene in onetissue while remaining largely “silent” in other tissue types. It willbe understood, however, that tissue-specific promoters may have adetectable amount of “background” or “base” activity in those tissueswhere they are silent. The degree to which a promoter is selectivelyactivated in a target tissue can be expressed as a selectivity ratio(activity in a target tissue/activity in a control tissue). In thisregard, a tissue specific promoter useful in the practice of thedisclosure typically has a selectivity ratio of greater than about 5.Preferably, the selectivity ratio is greater than about 15.

In certain indications, it may be desirable to activate transcription atspecific times after administration of the recombinant replicationcompetent retrovirus of the disclosure (RRCR). This may be done withpromoters that are hormone or cytokine regulatable. For example intherapeutic applications where the indication is a gonadal tissue wherespecific steroids are produced or routed to, use of androgen or estrogenregulated promoters may be advantageous. Such promoters that are hormoneregulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormoneregulated promoters such as those responsive to thyroid, pituitary andadrenal hormones may be used. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1990), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin. Tumor specific promoterssuch as osteocalcin, hypoxia-responsive element (HRE), MAGE-4, CEA,alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulategene expression in tumor cells.

In addition, this list of promoters should not be construed to beexhaustive or limiting, those of skill in the art will know of otherpromoters that may be used in conjunction with the promoters and methodsdisclosed herein.

TABLE 1 TISSUE SPECIFIC PROMOTERS Tissue Promoter Pancreas InsulinElastin Amylase pdr-1 pdx-1 glucokinase Liver Albumin PEPCK HBV enhancerα fetoprotein apolipoprotein C α-1 antitrypsin vitellogenin, NF-ABTransthyretin Skeletal muscle Myosin H chain Muscle creatine kinaseDystrophin Calpain p94 Skeletal alpha-actin fast troponin 1 Skin KeratinK6 Keratin K1 Lung CFTR Human cytokeratin 18 (K18) Pulmonary surfactantproteins A, B and C CC-10 P1 Smooth muscle sm22 α SM-alpha-actinEndothelium Endothelin-1 E-selectin von Willebrand factor TIE (Korhonenet al., 1995) KDR/flk-1 Melanocytes Tyrosinase Adipose tissueLipoprotein lipase (Zechner et al., 1988) Adipsin (Spiegelman et al. ,1989) acetyl-CoA carboxylase (Pape and Kim, 1989) glycerophosphatedehydrogenase (Dani et al., 1989) adipocyte P2 (Hunt et al., 1986)Breast Whey Acidic Protein (WAP) (Andres et al. PNAS 84: 1299-1303 1987Blood β-globin

It will be further understood that certain promoters, while notrestricted in activity to a single tissue type, may nevertheless showselectivity in that they may be active in one group of tissues, and lessactive or silent in another group. Such promoters are also termed“tissue specific”, and are contemplated for use with the disclosure. Forexample, promoters that are active in a variety of central nervoussystem (CNS) neurons may be therapeutically useful in protecting againstdamage due to stroke, which may affect any of a number of differentregions of the brain. Accordingly, the tissue-specific regulatoryelements used in the disclosure, have applicability to regulation of theheterologous proteins as well as applicability as a targetingpolynucleotide sequence in the present retroviral vectors.

In yet another embodiment, the disclosure provides plasmids comprising arecombinant retroviral derived construct. The plasmid can be directlyintroduced into a target cell or a cell culture such as NIH 3T3 or othertissue culture cells. The resulting cells release the retroviral vectorinto the culture medium.

The disclosure provides a polynucleotide construct comprising from 5′ to3′: a promoter or regulatory region useful for initiating transcription;a psi packaging signal; a gag encoding nucleic acid sequence, a polencoding nucleic acid sequence; an env encoding nucleic acid sequence;an internal ribosome entry site nucleic acid sequence; a heterologouspolynucleotide encoding a marker, therapeutic or diagnostic polypeptide;and a LTR nucleic acid sequence. As described elsewhere herein and asfollows the various segment of the polynucleotide construct of thedisclosure (e.g., a recombinant replication competent retroviralpolynucleotide) are engineered depending in part upon the desired hostcell, expression timing or amount, and the heterologous polynucleotide.A replication competent retroviral construct of the disclosure (e.g.,comprising SEQ ID NO:19, 20 or 22) can be divided up into a number ofdomains that may be individually modified by those of skill in the art.

For example, the promoter can comprise a CMV promoter having a sequenceas set forth in SEQ ID NO:19, 20 or 22 from nucleotide 1 to aboutnucleotide 582 and may include modification to one or more (e.g., 2-5,5-10, 10-20, 20-30, 30-50, 50-100 or more nucleic acid bases) so long asthe modified promoter is capable of directing and initiatingtranscription. In one embodiment, the promoter or regulatory regioncomprises a CMV-R-U5 domain polynucleotide. The CMV-R-U5 domaincomprises the immediately early promoter from human cytomegalovirus tothe MLV R-U5 region. In one embodiment, the CMV-R-U5 domainpolynucleotide comprises a sequence as set forth in SEQ ID NO:19, 20 or22 from about nucleotide 1 to about nucleotide 1202 or sequences thatare at least 95% identical to a sequence as set forth in SEQ ID NO:19,20, or 22 wherein the polynucleotide promotes transcription of a nucleicacid molecule operably linked thereto. The gag domain of thepolynucleotide may be derived from any number of retroviruses, but willtypically be derived from an oncoretrovirus and more particularly from amammalian oncoretrovirus. In one embodiment the gag domain comprises asequence from about nucleotide number 1203 to about nucleotide 2819 or asequence having at least 95%, 98%, 99% or 99.8% (rounded to the nearest10^(th)) identity thereto. The pol domain of the polynucleotide may bederived from any number of retroviruses, but will typically be derivedfrom an oncoretrovirus and more particularly from a mammalianoncoretrovirus. In one embodiment the pol domain comprises a sequencefrom about nucleotide number 2820 to about nucleotide 6358 or a sequencehaving at least 95%, 98%, 99% or 99.9% (roundest to the nearest 10^(th))identity thereto. The env domain of the polynucleotide may be derivedfrom any number of retroviruses, but will typically be derived from anoncoretrovirus or gamma-retrovirus and more particularly from amammalian oncoretrovirus or gamma-retrovirus. In some embodiments theenv coding domain comprises an amphotropic env domain. In one embodimentthe env domain comprises a sequence from about nucleotide number 6359 toabout nucleotide 8323 or a sequence having at least 95%, 98%, 99% or99.8% (roundest to the nearest 10^(th)) identity thereto. The IRESdomain of the polynucleotide may be obtained from any number of internalribosome entry sites. In one embodiment, IRES is derived from anencephalomyocarditis virus. In one embodiment the IRES domain comprisesa sequence from about nucleotide number 8327 to about nucleotide 8876 ora sequence having at least 95%, 98%, or 99% (roundest to the nearest10^(th)) identity thereto so long as the domain allows for entry of aribosome. The heterologous domain can comprise a cytosine deaminase ofthe disclosure. In one embodiment, the CD polynucleotide comprises ahuman codon optimized sequence. In yet another embodiment, the CDpolynucleotide encodes a mutant polypeptide having cytosine deaminase,wherein the mutations confer increased thermal stabilization thatincrease the melting temperature (T_(m)) by 10° C. allowing sustainedkinetic activity over a broader temperature range and increasedaccumulated levels of protein. In one embodiment, the cytosine deaminasecomprises a sequence as set forth in SEQ ID NO:19 or 22 from aboutnucleotide number 8877 to about 9353. The heterologous domain may befollowed by a polypurine rich domain. The 3′ LTR can be derived from anynumber of retroviruses, typically an oncoretrovirus and preferably amammalian oncoretrovirus. In one embodiment, the 3′ LTR comprises aU3-R-U5 domain. In yet another embodiment the LTR comprises a sequenceas set forth in SEQ ID NO:19 or 22 from about nucleotide 9405 to about9998 or a sequence that is at least 95%, 98% or 99.5% (rounded to thenearest 10^(th)) identical thereto.

The disclosure also provides a recombinant retroviral vector comprisingfrom 5′ to 3′ a CMV-R-U5, fusion of the immediate early promoter fromhuman cytomegalovirus to the MLV R-U5 region; a PBS, primer binding sitefor reverse transcriptase; a 5′ splice site; a psi (ψ) packaging signal;a gag, ORF for MLV group specific antigen; a pol, ORF for MLV polymerasepolyprotein; a 3′ splice site; a 4070A env, ORF for envelope protein ofMLV strain 4070A; an IRES, internal ribosome entry site ofencephalomyocarditis virus; a modified cytosine deaminase(thermostablized and codon optimized); a PPT, polypurine tract; and aU3-R-U5, MLV long terminal repeat. This structure is further depicted inFIG. 3.

The disclosure also provides a retroviral vector comprising a sequenceas set forth in SEQ ID NO:19, 20 or 22.

The retroviral vectors can be used to treat a wide range of disease anddisorders including a number of cell proliferative diseases anddisorders (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764; Friedmann,1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, R.Crystal, 1995, Science 270:404-410, each of which are incorporatedherein by reference in their entirety, see also, The Development ofHuman Gene Therapy, Theodore Friedmann, Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN 0-87969-528-5,which is incorporated herein by reference in its entirety).

The disclosure also provides gene therapy for the treatment of cellproliferative disorders. Such therapy would achieve its therapeuticeffect by introduction of an appropriate therapeutic polynucleotide(e.g., antisense, ribozymes, suicide genes, siRNA), into cells ofsubject having the proliferative disorder. Delivery of polynucleotideconstructs can be achieved using the recombinant retroviral vector ofthe disclosure, particularly if it is based on MLV, which is capable ofinfecting dividing cells.

In addition, the therapeutic methods (e.g., the gene therapy or genedelivery methods) as described herein can be performed in vivo or exvivo. It may be preferable to remove the majority of a tumor prior togene therapy, for example surgically or by radiation. In some aspects,the retroviral therapy may be preceded or followed by surgery,chemotherapy or radiation therapy.

The methods and compositions of the disclosure are useful in combinationtherapies including therapies with bevacizumab. As described herein areplication competent retrovirus (RCR) of the disclosure comprising atherapeutic (e.g., a cytotoxic gene) is useful in treating cellproliferative disorders. An advantage of the RCR of the disclosureincludes its ability to infect replicating cells cancer cells. Where thetransgene of the vector comprises a cytotoxic gene (e.g., a gene thatencodes a polypeptide that converts a non-cytotoxic agent to a cytotoxicagent) provides the ability to kill cancer cells.

In another embodiment, the methods and composition of the disclosure areuseful in combination with agents that promote apoptosis or that modifyexpression of cytokines or agents that promote apoptosis. For example, aretroviral vector of the disclosure comprising a polynucleotide encodinga polypeptide having cytosine deaminase activity can be administeredprior to, simultaneously with, or after administration of a peptide orpolypeptide having thymosin-alpha-1 activity. In one embodiment, thethymosin-alpha-1 polypeptide is administered at about 0.1-16 mg/kg.

Thymosin alpha-1 (Zadaxin™) functions by increasing the sensitivity ofneoplastic cells to chemotherapeutic agents by upregulatingpro-apoptotic proteins. Specifically, Thymosin alpha-1 upregulatespro-apoptotic FasL, FasR and TNFalpha-R1. In combination with a RCR ofthe disclosure, Thymosin alpha-1 functions as an adjuvant to increasethe sensitivity of neoplastic cells to 5-FU thereby increasing theeffectiveness of Toca 511 5-FC to 5-FU conversion as a chemotherapeuticagent after administration of RCR derived from T5.0002 and known as Toca511. Thymosin alpha-1 can also function as an immunomodulatory agentincreasing the recruitment and activity of immune components therebyleading to enhancement of vaccine effectiveness of RRV therapy.

A polypeptide having thymosin-alpha-1 activity refers to a polypeptidecomprising thymosin-alpha-1 or a variant or homolog thereof.Thymosin-alpha-1 (TA1) is a 28-amino acid peptide and includes syntheticforms of a naturally occurring hormone that circulates in the thymus.TA1 stimulate thymocyte growth and differentiation, production of IL-2,T cell IL-2 receptors, IFN-γ and IFN-α. Dosing regimes for TA1 are wellknown. In any case doses in humans can be over a wide range such as 1 to100 mg/dose.

The disclosure thus provides administering alpha thymosin peptides(“thymosin peptides”) to enhance cancer therapy with a replicationcompetent retroviral vector of the disclosure comprising heterologousgene encoding a polypeptide having cytosine deaminase activity. Thymosinpeptides include thymosin alpha 1 (“TA1”), and peptides havingstructural homology to TA1. TA1 is a peptide having the amino acidsequenceSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-11e-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn(SEQ ID NO:73) The amino acid sequence of TA1 is disclosed in U.S. Pat.No. 4,079,137, the disclosure of which is hereby incorporated byreference. TA1 is a non-glycosylated 28-amino acid peptide having anacetylated N-terminus, and a molecular weight of about 3108. A syntheticversion of TA1 is commercially available in certain countries under thetrade name ZADAXIN®.

It is believed that thymosin peptides (e.g., TA1), among other things,activate Toll-like Receptor 9 (TLR), resulting in increases in Th1cells, B cells, and NK cells, thereby leading to enhancement of vaccineeffectiveness. For example, TA1 may increase or enhance lymphocyticinfiltration, secretion of chemotactic cytokines, maturation anddifferentiation of dendritic cells, secretion of thymopoeitic cytokinesincluding IFN-alpha, IL-7, and IL-15, and B cell production ofantibodies.

The thymosin peptides that find use with the vectors and methods of thedisclosure include naturally occurring TA1 (e.g., TA1 purified orisolated from tissues), as well as synthetic TA1 and recombinant TA1. Insome embodiments, the thymosin peptide comprises the amino acid sequenceof SEQ ID NO:73 (where an acylated, e.g., acetylated, N-terminus isoptional). In some embodiments, the thymosin peptide comprises an aminoacid sequence that is substantially similar to TA1, and maintains theimmunomodulatory activity of TA1. The substantially similar sequence mayhave, for example, from about 1 to about 10 amino acid deletions,insertions, and/or substitutions (collectively) with respect to TA1. Forexample, the thymosin peptide may have from about 1 to about 5 (e.g., 1,2, or 3) amino acid insertions, deletions, and/or substitutions(collectively) with respect to TA1 so long as the peptide has one ormore activities associated with a naturally occurring thymosin.

Thus, a thymosin peptide useful in the methods of the disclosure maycomprise an abbreviated TA1 sequence, for example, having deletions offrom 1 to about 10 amino acids, or from about 1 to 5 amino acids, or 1,2 or 3 amino acids with respect to TA1. Such deletions may be at the N-or C-terminus, and/or internal, so long as the activity of the peptideis substantially maintained. Alternatively, or in addition, thesubstantially similar sequence may have from about 1 to about 5 aminoacid insertions (e.g., 1, 2, or 3 amino acid insertions) with respect toTA1, where the immunomodulatory activity of TA1 is substantiallymaintained. Alternatively, or in addition, the substantially similarsequence may have from 1 to about 10 amino acid substitutions, where theimmunomodulatory activity is substantially maintained. For example, thesubstantially similar sequence may have from 1 to about 5, or 1, 2, or 3amino acid substitutions, which may include conservative andnon-conservative substitutions. In some embodiments, the substitutionsare conservative. Generally, conservative substitutions includesubstitutions of a chemically similar amino acid (e.g., polar,non-polar, or charged). Substituted amino acids may be selected from thestandard 20 amino acids or may be a non-standard amino acid (e.g., aconserved non-standard amino acid).

In some embodiments, the thymosin peptide comprises an amino acidsequence having at least 70% sequence identity to SEQ ID NO:73, whilemaintaining the activity of a naturally occurring TA1. For example, thethymosin peptide may comprise an amino acid sequence having at least80%, 90%, or 95% sequence identity to SEQ ID NO:73. The thymosin peptidemay comprise an amino acid sequence having 100% sequence identity to SEQID NO:73. In all cases, the N-terminus may be optionally acylated (e.g.,acetylated) or alkylated, for example, with a C1-10 or C1-C7 acyl oralkyl group.

The disclosure provides methods for treating cell proliferativedisorders such as cancer and neoplasms comprising administering an RCRvector of the disclosure prior to, simultaneously with or followingadministration of a thymosin peptide. In another embodiment thecombination of RCR and thymosin may also be followed by treatment with achemotherapeutic agent or anti-cancer agent. In one aspect, the RCRvector is administered to a subject for a period of time prior toadministration of the chemotherapeutic or anti-cancer agent that allowsthe RCR to infect and replicate. The subject is then treated with achemotherapeutic agent or anti-cancer agent for a period of time anddosage to reduce proliferation or kill the cancer cells. In one aspect,if the treatment with the chemotherapeutic or anti-cancer agent reduces,but does not kill the cancer/tumor (e.g., partial remission or temporaryremission), the subject may then be treated with a non-toxic therapeuticagent (e.g., 5-FC) that is converted to a toxic therapeutic agent incells expression a cytotoxic gene (e.g., cytosine deaminase) from theRCR. The methods and compositions of the disclosure are useful in othercombination therapies, for example, therapies with Thymosin alpha-1(Zadaxin™), trastuzumab (Herceptin), Leucovorin and other folic acidanalogues, or other promoters of 5-FU activity (D. Papamichael StemCells 18:166-175 2000) such as dihydropyrimidine dehydrogenase [DPD]inhibitors [e.g. 5-Chloro-2,4-Dihydroxypyridine—Cdhp]) whose action istargeted, rather than systemic, when used in conjunction with the tumortargeted 5-FU production from 5-FC administration and CD expression fromthe vector of disclosure.

Leucovorin or other folic acid analogues promote 5-FU binding tothymidilate synthase, thereby inactivating this key enzyme in nucleicacid biosynthesis, and enhancing the efficacy of 5-FU.

DPD inhibitors block the activity of dihydropyrimdine dehydrogenaseanenzyme that normally degrades about 80% of systemically administered5-FU. DPD inhibition results in increased retention of 5-FU andfrequently make 5-FU very much more toxic. In fact this can be lifethreatening in patients that have DPD deficiency (Ezeldin & DiasioClinical Colorectal Cancer, Vol. 4, No. 3, 181-189, 2004). However, inthe vectors of the disclosure, 5-FU is only produced locally in thetumor, and hence the increased toxicity is confined to the area of thetumor, where it is a benefit.

The disclosure provides a method of treating a subject having a cellproliferative disorder. The subject can be any mammal, and is preferablya human. The subject is contacted with a recombinant replicationcompetent retroviral vector of the disclosure. The contacting can be invivo or ex vivo. Methods of administering the retroviral vector of thedisclosure are known in the art and include, for example, systemicadministration, topical administration, intraperitoneal administration,intra-muscular administration, intracranial, cerebrospinal, as well asadministration directly at the site of a tumor or cell-proliferativedisorder. Other routes of administration are known in the art.

Thus, the disclosure includes various pharmaceutical compositions usefulfor treating a cell proliferative disorder. The pharmaceuticalcompositions according to the disclosure are prepared by bringing aretroviral vector containing a heterologous polynucleotide sequenceuseful in treating or modulating a cell proliferative disorder accordingto the disclosure into a form suitable for administration to a subjectusing carriers, excipients and additives or auxiliaries. Frequently usedcarriers or auxiliaries include magnesium carbonate, titanium dioxide,lactose, mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

For example, and not by way of limitation, a retroviral vector useful intreating a cell proliferative disorder will include an amphotropic ENVprotein, GAG, and POL proteins, a promoter sequence in the U3 regionretroviral genome, and all cis-acting sequence necessary forreplication, packaging and integration of the retroviral genome into thetarget cell.

The following Examples are intended to illustrate, but not to limit thedisclosure. While such Examples are typical of those that might be used,other procedures known to those skilled in the art may alternatively beutilized.

EXAMPLES Example 1 Modification of Vector Backbone of pACE-GFPemd topAC3-GFPemd and Insertion of Cytosine Deaminase Gene Sequences in Placeof GFP

The previous back bone of the pACE-GFPemd plasmid (U.S. Pat. No.6,899,871, Wang et al. Hum Gene Ther 14:117 2003) was modified in 3 waysas shown in FIG. 3E. The modifications were made by PCR-mediated,oligonucleotide-directed mutagenesis (Logg et al., J. Mol Biol 369:1214, 2007; see also “Molecular Biology and Biotechnology” Eds. J M.Walker, R. Rapley, Royal Society of Chemistry, London UK, 2000). Thefollowing modifications were made. 1) The nucleic acid sequence at thep15 region at the 3′ end of the amphotropic env gene was originallyderived from the ecotropic envelope—this sequence was replaced by thecorresponding sequence from the 4070A amphotropic envelope; the encodedenvelope amino acids are identical in the two constructs. 2) The IRESsequence 3′ end was modified to allow easier insertion of transgenes ofchoice with insertion of a PstI1 site and small imperfect repeats ateither end of the IRES transgene site were removed. 3) Residual viralsequence downstream of the 3′LTR was removed. The resultant plasmid ispACE-emdGFP (aka pACE-GFP, pACE-eGFP and T5.0006) was used as a basisfor the vectors encoding cytosine deaminase and variants. Two methods ofinserting the coding sequence cassettes were used initially. The firstmethod resulted in the sequence 5′TTATAAT3′ (SEQ ID NO:74), and thesecond in the sequence 5′TTATAA3′(SEQ ID NO:75) immediately upstream ofthe ATG start codon. The second method was simpler, as it involvedsimple PstI1 and Not1 enzyme cuts in the vector and the syntheticcytosine deaminase genes, followed by religation. Vectors with cytosinedeaminase inserts were made both ways with the CDopt (CD1) and theCDopt+3pt (CD2) (see FIG. 2) coding sequences and infectious virus prepsmade by transient transfection of 293T cells as described in Example 3.U87 cells were then infected in culture, at an MOI of 0.1, and the cellsgrown until 100% infected. Cell extracts of 100% infected cells wereassayed for cytosine deaminase activity as described in Example 6 andthe specific activity of the enzyme was found to be equivalent forconstructs with either upstream sequence, that were otherwise identical.Therefore in the table in FIG. 2, pACE-eGFP (T5.0006) and pACE-yCD(T5.0007) have the first upstream sequence, while all other constructsthat were further tested have the second. Subsequently vectors withdifferent gene inserts have been routinely constructed withstraightforward PStI1 and Not I cuts.

See FIG. 3A below for a diagram of the vector construct for the initialtransfected replication-competent retrovirus. CMV is the human CMVimmediate early promoter, U3, R and U5 are the corresponding regions ofthe viral long terminal repeat (LTR). Gag, pol and env are the viralprotein coding regions. FIGS. 3B and 3D shows the plasmid structure anda sequence of the disclosure.

The vector of the disclosure provides a number of differences comparedto the vector of Tai et al., Mol. Ther. 12:842, 2005. The Tai et al.vector has been altered to eliminate about 70 bp of MLV sequencedownstream from the 3′LTR. The DNA sequence downstream of the ClaI sitein the envelope was changed to an amphotropic envelope sequence. Thischange does not change the amino acid sequence of the envelope. Inaddition, small repeats on either side of the IRES-CD cassette have beeneliminated to avoid instability due to homologous recombination. Thesechanges also unexpectedly provided increased stability of the vectorduring replication and passaging in host cells (FIG. 5).

It is recognized that after reverse transcription and the firstintegration event into treated cells, the DNA provirus and anysubsequent progeny retrovirus has a conventional LTR structure from MLVon either end. This configuration has been shown to be stable aftermultiple cycles of infection (See FIG. 5 below).

Example 2 Genetic Enhancements to the Wild Type Yeast Cytosine DeaminaseGene

Two sets of changes have been made: (1) three positional mutations whichchange three amino acids (A23L, I140L and V108I) to increase thermalstability of the yeast cytosine deaminase protein and (2) additionalgene sequence modifications to enhance human codon usage sequences toimprove protein translation efficiency in human cells without furtherchanges to the amino acid sequence.

Sequence design for CD included CD-optimized, CD-UPRT (+/− linker) andCD-OPRTase (+/− linker). The final cytosine deaminase coding sequencecan comprise at the 5′ end a PSI1 site (full length) and 3′ end NotIsite plus poly A tail for PSI1/Not1 cassette based strategy. Sequencescassettes were ordered from, and provided by, a commercial vendor(BioBasic Inc., Ontario, Canada).

The following sequence comprising a yeast cytosine deaminase was usedfor cloning, optimizing and mutation (the boxed nucleic acids comprisethe restriction sites—PsiI and NotI—used in subsequent methods forcloning:

(SEQ ID NO: 43)

TATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGATTGG

AAAAGGGGGGThe following Table summarizes the genes and resulting plasmid vectorsthat were made and their names.

TABLE Vector constructs and names Identity Reference Original 5′LTRTrans- Code name Name Prom Envelope Vector IRES gene 3′LTR T5.0000pACE-yCD pACE-CD CMV Ampho pACE EMCV Wt yeast MLV U3 (Tai et al. (4070A)CD 2005) T5.0001 pAC3-yCD1 CDopt CMV Ampho pAC3 EMCV modified MLV U3sequence (4070A) CD T5.0002 pAC3-yCD2 CDopt+3pt CMV Ampho pAC3 EMCVModified MLV U3 (4070A) CD T5.0003 pAC3-yCD2-U Cdopt+3pt- CMV Ampho pAC3EMCV CD2- MLV U3 UPRT (4070A) UPRT T5.0004 pAC3-yCD2-O CDopt+3pt- CMVAmpho pAC3 EMCV CD2- MLV U3 OPRT (4070A) OPRT T5.0005 pAC3-yCD2-LOCDopt+3pt- CMV Ampho pAC3 EMCV CD2-L- MLV U3 LINK-OPRT (4070A) OPRTT5.0006 pAC3-eGFP pAC3-emd, CMV Ampho pAC3 EMCV Emerald MLV U3 pAC3GFP(4070A) GFP T5.0007 pAC3-yCD pAC3-yCD CMV Ampho pAC3 EMCV Wt yeast MLVU3 (4070A) CD

The replication competent retroviral vector described by Kasahara et al.pACE-CD (U.S. Pat. No. 6,899,871, the disclosure of which isincorporated herein) was used as a basis for additional modifications. Avector (pAC3-yCD) was modified to express a modified yeast cytosinedeaminase gene as described herein and was used in the constructs. SeeFIG. 3A below for a diagram of the vector construct for the initialtransfected replication-competent retrovirus. CMV is the human CMVimmediate early promoter, U3, R and U5 are the corresponding regions ofthe viral long terminal repeat (LTR). Gag, pol and env are the viralprotein coding regions. FIGS. 3B and 3D shows the plasmid structure anda sequence of the disclosure.

After the genes were synthesized at a contractor (Bio Basic Inc.,Markham, Ontario, Canada) they were inserted into the Psi1-Not1 site ofthe pAC3 vector backbone (FIG. 3). The plasmid backbone was normallygenerated by cutting the plasmid pAC3-eGFP with PsiI and NotI andpurifying the large (about 11 kb) fragment encoding the plasmid andretroviral backbone)

A. Humanized Codon Optimized CD Gene (CD-Opt, Aka CD1, T5.0001).

A comparison of a human codon optimized cytosine deaminase of Conrad etal. and PCT WO 99/60008 indicates 91 total codons optimized in both, 36codons identical, 47 codons had third base pair changes (all encode sameamino acid) and 9 codons were different (however they encoded same aminoacid). Of the 9 codons that differed:

AGC (Ser) to TCC (Ser) CGT (Arg) to AGG (Arg) CCA (Pro) to CCT (Pro)

All have equivalent GC content and encode the same amino acid. Thenative yeast gene sequence above was separately codon optimized to givethe following CD gene (CD1) and was called T5.0001 when inserted intothe plasmid vector pAC3 which encodes the replication competentretrovirus (RCR) with IRES.

(SEQ ID NO: 44)

AGGCCGCCCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGGTGGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGATCATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGG

G.

B. Heat Stabilized CD Gene.

Additional modifications were made to enhance the stability of thecytosine deaminase. Genetic enhancements to the wild type yeast cytosinedeaminase gene were made to include three positional mutations whichchange three amino acids (A23L, I140L and V108I) to increase thermalstability of the yeast cytosine deaminase protein.

The following primer pairs were used in the generation of the gene forthe cytosine deaminase polypeptide of the disclosure:

Site directed mutagenesis primers: Primers sense: (SEQ ID NO: 45)5′-tcgaggatatcggcgagtgaaacccgttattctttttggc-3′ Primers antisense:(SEQ ID NO: 46) 5′-gccaaaaagaataacgggtttcactcgccgatatcctcga-3′Primers sense: (SEQ ID NO: 47)5′tcggcgagtgatccggcggcggcgcctccggcggcggcgcctccggcggcggcgcctccggcggcggcgccaacccgttatt-3′ Primers antisense: (SEQ ID NO: 48)5′-aataacgggttggcgccgccgccggaggcgccgccgccggaggcgccgccgccggaggcgccgccgccggatcactcgccga-3′

To increase the stability of the native yeast CD protein, three aminoacid substitutions were engineered into the protein. These substitutionswere alone or in combination with human codon optimization.

The three amino acid substitutions are: A23L, V108I, I140L. A sequenceencoding these substitutions is shown below.

(SEQ ID NO: 3)ATGGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCG T TATTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTC ATC GGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAG TTA ATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTG

The encoded polypeptide comprises the following sequence (substitutedamino acids in underlined):

(SEQ ID NO: 4)   1 MVTGGMASKWDQKGMDIAYEEA LLGYKEGGVPIGGCLINNKDGSVLGRGHNMRFQKGSAT  61LHGEISTLENCGRLEGKVYKDTTLYTTLSPCDMCTGAIIMYGIPRCV I GENVNFKSKGEK 121YLQTRGHEVVVVDDERCKK L MKQFIDERPQDWFEDIGE-

Final construct design that integrates 3 amino acid substitutionsA23L/V108I/I140L utilizing preferred codons and uses preferred humancodon usage for entire sequence (this gene is called CDopt+3pt [aka CD2]and T5.0002 when inserted into the plasmid vector pAC3 which encodes theRCR with IRES.

(SEQ ID NO: 49)   1ATGGTGACCGGCGGCATGGCCTCCAAGTGGGATCAAAAGGGCATGGATATCGCTTACGAG  61GAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAAC 121AAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACC 181CTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAG 241GACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATG 301TACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAG 361TACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTG 421ATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTGATAAUnderlined codons denote preferred codons for amino acid substitutions.

Protein translation sequence alignment indicates preferred codon changesand amino acid substitutions result in desired protein structure:

CD-optimized sequence design (human codon preference+3 amino acidsubstitutions)

(SEQ ID NO: 50)

TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG

AAAAGGGGGG

C. Construction of CD-UPRT Fusion Gene (CDopt+3pt-UPRT, [Aka CDopt-UPRTand CD2-UPRT], T5.0003 in the pAC3 Plasmid RCR Vector).

A fusion construct was also developed comprising a CD polypeptide asdescribed above linked to a UPRT polypeptide to generate aCD-optimized-UPRT. The following primers were used to delete thestop-start between the CD and UPRT.

Primer Sequences:

Primer Name Primer Sequence (5′ to 3′) (SEQ ID NO:) del118-1235′-tcgaggatatcggcgagtgaaacccgttattctttttggc-3′ (51) del118-123-antisense5′-gccaaaaagaataacgggtttcactcgccgatatcctcga-3′ (52) Energy Cost LengthDuplex Energy of Primer Name (nt.) Tm at 68° C. Mismatches del118-123 4079.06° C. −44.37 kcal/mole 21.1% del118-123-antisense 40 79.06° C.−47.95 kcal/mole 20.3% Primer Name Primer-Template Duplex del118-123(SEQ ID NOs: 51 and 53, respectively

del118-123-anti-sensense (SEQ ID NO: 54 and 52 respectively)

The resulting fusion polynucleotide comprises 1296 bp and the sequenceset forth immediately below:

(SEQ ID NO: 55)

TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGAACCCGTTATTCTTTTTGGCTTCTCCATTCTTGTACCTTACATATCTTATATATTATCCAAACAAAGGGTCTTTCGTTAGCAAACCTAGAAATCTGCAAAAAATGTCTTCGGAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTT

TTTAGTCTCCAGAAAAAGGGGGG 

D. Construction of CD-Linker UPRT Fusion Gene (CDopt+3pt-LINK-UPRT [AkaCDopt-LINKER-UPRT and CD2-L-UPRT].

A fusion construct was also developed by cloning a linker(Ser-Gly-Gly-Gly-Gly)₄ (SEQ ID NO:56) domain between and in frame withthe CD polypeptide and the UPRT polypeptide to generated aCD-optimized-linker-UPRT sequence. The following primers were used toinsert the linker.

Primer Name Primer Sequence (5′ to 3′)(SEQ ID NO:) ins_60nt_after_4775′- tcggcgagtgatccggcggcggcgcctccggcggcggcgcctccggcggcggcgcctccggcggcggcgccaacccgttatt-3′(57) ins_60nt_after_477- 5′-antisense aataacgggttggcgccgccgccggaggcgccgccgccggaggcgccgccgccggaggcgccgccgccggatcactcgccga-3′(58) Energy Cost LengthDuplex Energy at of Primer Name (nt.) Tm 68° C. Mismatchesins_60nt_after_477 82 79.77° C. −30.19 kcal/mole 83.3%ins_60nt_after_477- 82 79.77° C. −32.31 kcal/mole 82.2% antisense

The resulting construct has size: 1356 bp and the sequence immediatelybelow:

(SEQ ID NO: 59)

TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCAACCCGTTATTCTTTTTGGCTTCTCCATTCTTGTACCTTACATATCTTATATATTATCCAAACAAAGGGTCTTTCGTTAGCAAACCTAGAAATCTGCAAAAAATGTCTTCGGAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTTGGGTGAC

TCCAGAAAAAGGGGGG

E. Construction of CD-OPRT Fusion Gene (CDopt+3pt-OPRT [Aka CDopt-OPRTand CD2-OPRT], T5.0004 when Inserted into the pAC3 Plasmid RCR Vector).

A fusion construct was also developed comprising a CD polypeptide asdescribed above linked to an OPRT polypeptide to generate aCD-optimized-OPRTase (CD humanized+3ptmutation+OPRTase functional domainhuman).

The resulting construct comprises a size of 1269 bp and the sequenceimmediately below:

(SEQ ID NO: 60)

TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGGCGGTCGCTCGTGcagctttggggccattggtgacgggtctgtacgacgtgcaggctttcaagtttggggacttcgtgctgaagagcgggctttcctcccccatctacatcgatctgcggggcatcgtgtctcgaccgcgtcttctgagtcaggttgcagatattttattccaaactgcccaaaatgcaggcatcagttttgacaccgtgtgtggagtgccttatacagctttgccattggctacagttatctgttcaaccaatcaaattccaatgcttattagaaggaaagaaacaaaggattatggaactaagcgtcttgtagaaggaactattaatccaggagaaacctgtttaatcattgaagatgttgtcaccagtggatctagtgttttggaaactgttgaggttcttcagaaggagggcttgaaggtcactgatgccatagtgctgttggacagagagcagggaggcaaggacaagttgcaggcgcacgggatccgcctccactcagtgtgtacattgtccaaaatgctggagattctcgagcagcagaaaaaagttgatgctgagacagttgggagagtgaagaggtttattcaggagaatgtctttgtggcagcgaatcataatggttctcccctttctataaaggaagcacccaaagaactcaGCTTCGGTGCACGTGCAGAGCTGCCCAGGATCCACCCAGTTGCATC

F. Construction of CD-Linker-OPRT Fusion Gene (CDopt+3pt-LINK-OPRT, [AkaCDopt-LINKER-OPRT and CD2-L-OPRT], T5.0005 in the pAC3 plasmid RCRvector).

A fusion construct was also developed by cloning a linker(Ser-Gly-Gly-Gly-Gly)₄) (SEQ ID NO:56) domain between and in frame withthe CD polypeptide and the OPRT polypeptide to generated aCD-optimized-linker-OPRT sequence.

The resulting construct comprises a size of 1329 bp and the sequenceimmediately below:

(SEQ ID NO: 61)

TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCGCGGTCGCTCGTGcagctttggggccattggtgacgggtctgtacgacgtgcaggctttcaagtttggggacttcgtgctgaagagcgggctttcctcccccatctacatcgatctgcggggcatcgtgtctcgaccgcgtcttctgagtcaggttgcagatattttattccaaactgcccaaaatgcaggcatcagttttgacaccgtgtgtggagtgccttatacagctttgccattggctacagttatctgttcaaccaatcaaattccaatgcttattagaaggaaagaaacaaaggattatggaactaagcgtcttgtagaaggaactattaatccaggagaaacctgtttaatcattgaagatgttgtcaccagtggatctagtgttttggaaactgttgaggttcttcagaaggagggcttgaaggtcactgatgccatagtgctgttggacagagagcagggaggcaaggacaagttgcaggcgcacgggatccgcctccactcagtgtgtacattgtccaaaatgctggagattctcgagcagcagaaaaaagttgatgctgagacagttgggagagtgaagaggtttattcaggagaatgtctttgtggcagcgaatcataatggttctcccctttctataaaggaagcacccaaagaactcaGCTTCGGTGCACGTGCAGAGCTGCCCAGGATCCACCCAGTTGCATCGAAGTAA

FIG. 4 demonstrates that higher levels of the human codon optimized withthe three mutations for higher stability are observed compared to wildtype yCD protein in infected U-87 cells.

Example 3 Vector Production by Transient Transfection

Vector can be produced in a number of ways, but the first step is tointroduce the DNA vector into cells to allow production of infectiousparticles, that can then be harvested from the cell supernatant. Onceinfectious particles have been generated other methods of production canbe implemented by those skilled in the art. Vector particles weregenerated by transient transfection of 293T cells (Pear et al. Proc NatlAcad Sci USA. 90:8392-8396 1993).

The 293T cells were thawed and put into culture, then passaged twice inT-75 flasks containing 15 mL of the DMEM medium that was prepared bymixing DMEM High Glucose medium (Hyclone#30081, 500 mL) with FBS(Hyclone# SH30070, 50 mL), L-Glutamine (Cellgro#25-005-CI, 5 mL), NEAA(Hyclone #SH30238, 5 mL), and Penicillin-strep (Cellgro#30-002-CI, 5mL). The flasks were incubated at 37° C. and 5% CO₂. After the 3^(rd)passage cells were seeded in 6 T-25's, each containing 5 mL of themedium, at a cell density of 1.8×10⁶ cells/T-25 (or 7.2×10⁴ cells/cm²).One day after seeding the T-25's, the cells were transfected with theT5.0002 plasmid that expressed the viral vector using the CalciumPhosphate Transfection Kit from Promega (Cat# E1200). Eighteen hoursfollowing transfection, the media in one set of the flasks (3 flaskseach set) were replaced with fresh medium containing 10 mM NaB. Themedia in the 2^(nd) set of the flasks were not replaced, which served asa control (zero NaB). Eight hours post NaB treatment the media in allflasks were replaced with the fresh medium containing no NaB. Theexpression was allowed to continue for both sets of flasks until thenext day (22 hours duration). The supernatants from both sets of flaskswere harvested and assayed for their titers by qPCR expressed inTransducing Units (TU)/ml (see Example 4).

The titer results are shown in the following table.

Second titer (after storing Condition First titer at −80° C. for 68days) Without NaB 1.5 (±0.05) × 10⁶ TU/mL 1.2 (±0.2) × 10⁶ TU/mL 10 mMNaB 1.4 (±0.3) × 10⁶ TU/mL 7.0 (±0.14) × 10⁵ TU/mL TU = transductionunit

Subsequent vector preparations were produced in this manner, withoutsodium butyrate. Other vector plasmids (Table 2) have been used in thesame way to generate vector preparations with titers between 1E5 TU/mland 1E7 TU/ml. Such material can be further purified and concentrated,if desired, as described below see also: U.S. Pat. No. 5,792,643; T.Rodrigues et al. J Gene Med 9:233 2007.

In certain embodiments of the disclosure the dosing was calculated bygrams of brain weight. In such embodiments, the dosing of a replicationcompetent retroviral vector of the disclosure useful in the methods fortreatment can range from 10⁴ to 10⁶ TU per gram brain weight.

Example 4 Quantitative PCR Titering Assay

The functional vector concentration, or titer, is determined using aquantitative PCR-based (qPCR) method. In this method, vector is titeredby infecting a transducible host cell line (e.g. PC-3 human prostaticcarcinoma cells, ATCC Cat# CRL-1435) with a standard volume of vectorand measuring the resulting amount of provirus present within the hostcells after transduction. The cells and vector are incubated understandard culturing condition (37° C., 5% CO₂) for 24 hr to allow forcomplete infection prior to the addition of the anti-retroviral AZT tostop vector replication. Next, the cells are harvested from the culturedish and the genomic DNA (gDNA) is purified using an Invitrogen PurelinkgDNA purification kit and eluted from the purification column withsterile RNase-/DNase-free water. The A₂₆₀/A₂₈₀ absorbance ratio ismeasured on a spectrophotometer to determine the concentration andrelative purity of the sample. The gDNA concentrations are normalizedwith additional RNase-/DNase-free water to the lowest concentration ofany given set of gDNA preparations such that the input DNA for the qPCRis constant for all samples analyzed. Genomic DNA purity is furtherassessed by electrophoresis of an aliquot of each sample on an ethidiumbromide stained 0.8% agarose gel. If the sample passes an A₂₆₀/A₂₈₀absorbance range of 1.8-2.0 and shows a single band of gDNA, then thesample is ready for qPCR analysis of provirus copy number of the vector.Using primers that interrogate the LTR region of the provirus(reverse-transcribed vector DNA and vector DNA that is integrated intothe host gDNA), qPCR is performed to estimate the total number oftransduction events that occurred when the known volume of vector wasused to transduce the known number of cells. The number of transductionevents per reaction is calculated from a standard curve that utilizes atarget-carrying plasmid of known copy-number that is serial diluted from10⁷ to 10 copies and measured under identical qPCR conditions as thesamples. Knowing how many genomic equivalents were used for each qPCRreaction (from the concentration previously determined) and how manytransduction events that occurred per reaction, we determine the totalnumber of transduction events that occurred based on the total number ofcells that were present at the time of transduction. This value is thetiter of the vector after dilution into the medium containing the cellsduring the initial transduction. To calculate the corrected titer value,the dilution is corrected for by multiplying through by the volume ofculture and the volume of titer divided by the volume of titer. Theseexperiments are performed in replicate cultures and analyzed by qPCRusing triplicate measurements for each condition to determine an averagetiter and with its associated standard deviation and coefficient ofvariance.

Example 5 Vector Testing

In order to be effective vector constructs and their derived infectiousparticles need to: (1) make good titer of virus by transienttransfection (see Examples 3 and 4); (2) be stable upon multiplepassages; (3) kill cells efficiently in the presence of 5-FC; and (4)express enzyme activity upon infection of target cells. Example 3 showsthat useful titers can be obtained from the vectors.

Genetic Stability of Viral Vectors.

To demonstrate the stability the following experiment was performed.Approximately 10⁶ naïve U-87 cells were initially infected with theviral vector at an MOI of 0.01, and grown until fully infected tocomplete a single cycle of infection. Supernatant is then repassed ontouninfected cells and the cycle repeated. In this experiment, twelvecycles have been completed in duplicate trials (FIG. 5 shows one of eachof the duplicate trials; the other duplicates gave similar results).Genomic stability of the yCD2 or other transgene sequence is assessed byPCR amplification of the integrated provirus from the infected cellsusing MLV specific primers flanking the transgene insertion site. Theappearance of any bands smaller than full-length amplicon would be anindicator of vector instability. FIG. 5 demonstrates that a vector ofthe disclosure (T5.0007-comprising the modified vector and CDheterologous polynucleotide) maintains stability for more passages thanpACE-CD. Furthermore T5.0003 is somewhat less stable while T5.0004 andT5 appear about as stable as pACE-CD. pACE-CD has been used in mousetumor studies and shows good anti-tumor effects in mouse models. Howevera more stable viral genome will be much more potent and long lasting intreatment of animals and humans, especially if multiple cycles of 5-FCtreatment are required. Both T5.0001 and T5.0002 are markedly morestable than even T5.0007, as shown by the reduced presence of smallbands at later passages (FIG. 5), showing that silent changes in aprotein coding sequence or small changes that result in point mutationscan lead to unexpectedly superior properties with more stable vectorgenomes.

Cell Killing Experiments.

The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) isa colorimetric method for determining the number of viable cells inproliferation assays. We have utilized this assay to determine cellgrowth kinetics, as well as to determine the dose response of variouscell lines to 5-Fluorocytosine (5-FC) and 5-Fluorouracil (5-FU).

Cells 100% infected with vector were seeded at 1000 cells/well in96-well plates. They were monitored over an eight day period followingtreatment with various concentrations of 5-FC (5-FU for controls). Ananalysis of their cell growth was assessed every two days utilizingPromega's Cell Titer 96 AQueous One Solution reagent (MTS). Briefly, 20μl of MTS was mixed with 100 μl media (as recommended by themanufacturer) and added to the samples in the 96-well plate. The sampleswere incubated for 60 minutes in a 37° C./5% CO₂ incubator. Thereafter,absorbance readings were taken on a plate reader at a 490 nm wavelength.

FIG. 6A shows the results of an experiment that demonstrates that thecytosine deaminase in cells expressing the yCD2 protein is at least asactive as that from cells expressing the wild type yCD protein, byperforming 5-FC titrations on U-87 cells infected either with AC3-yCD2(vector) or AC3-yCD (vector). Briefly, U-87 cells 5 days post infectionat a multiplicity of infection of 0.1 (i.e. 100% infected) with eitherAC3-yCD (wild type CD) vector or AC3-yCD2 (thermostabilized & codonoptimized) vector were subject to increasing amounts of 5-FC or 0.1 mMof 5-FU as a positive control for 8 days. On day 8, cell cultures wereassessed for viability using an MTS assay (Promega CellTiter 96 AQUEOUSOne Solution Proliferation Assay). Data shows comparable killing betweenthe two retroviral vectors at increasing doses of 5-FC treatment.

In similar in-vitro cell culture experiments with RG2 cells (ATCC Cat#CRL-2433), the RG2 cell line was transduced with 5 different vectors(pACE-CD, T5.0001, T5.0002, T5.0004, and T5.0007). It was subsequentlysubject to increasing concentrations of 5-FC (5-FU for controls) for 8days and monitored as described above. The results are shown in FIG. 2.Concentrations of 0.01 mM were sufficient to induce complete killingwith all vectors tested with the exception of wild type-yeast CytosineDeaminase (pACE-yCD). It was less sensitive and required 1.0 mM of 5-FCfor complete killing.

CD Expression Assay.

U87 cells were transduced at a multiplicity of infection (MOI) of 0.1,cultivated for 5 days to allow viral spread and cells from day 5 posttransduction were harvested. The cells were then collected bycentrifugation at 800×g for 5 min. The supernatant was aspirated awayfrom the cell pellet and washed with 5 mL of phosphate buffered saline(PBS) and again centrifuged at 800×g for 5 min. The resulting cellpellet was taken up in 1.5 mL of PBS, resuspended by passage through apipette tip and placed in a freezer at −20° C. Cells were lysed by afreeze/thaw method. Previously resuspended cells were allowed to thaw atroom temperature, passed through a pipette tip, mixed with proteaseinhibitor cocktail and again refrozen at −20° C. Previous to the enzymeassay, the sample was again thawed at room temperature and passedthrough a pipette tip. The suspension was then centrifuged at 14,000 rpmin a tabletop centrifuge for 5 min. The supernatant was decanted awayfrom the pellet and placed in a fresh eppendorf tube and placed on ice.

yCD enzyme activity was assessed by using an HPLC assay. The HPLC assaywas performed on a Shimadzu LC20AT unit connected in series with aphotoarray detector and autoinjector. The solid phase was a Hypersil BDSC₁₈, HPLC column with a 5 μm sphere size and 4.0×250 mm columndimensions. The mobile phase was 50 mM ammonium phosphate, pH 2.1,containing 0.01% tert-butylammonium perchlorate and 5% methanol; thesystem was equilibrated at 22° C. All reagents were ACS grade andsolvents were HPLC grade. A reaction mix was made consisting of 800 μLwith a final concentration of 0.125 mg/mL 5-FC (1 mM) in PBS and placedin a 1.5 mL autosampler vial. The reaction was then initiated by adding200 μL of each cell lysate. The reaction/autosampler vials were placedin the auto sampler and 5 μL of the reaction mixture was injected. Timepoints were taken periodically by retrieving a 5 μL aliquot from eachreaction vial and analyzing on the HPLC column. The conversion rates of5-FC to 5-FU were calculated by comparing the peak areas with knownamounts from a previously generated standard curve of 5-FU. The rate of5-FC conversion to 5-FU was derived by plotting the amount of 5-FU (innmol) generated against its corresponding time interval. Proteinconcentration for the cell sample was derived and the Specific Activityof the cell lysate samples were calculated by dividing the conversionrate (nmol/min) by the amount of protein used in the assay in mg.

FIG. 6B shows the specific activity of various vectors after 5 days ontransduction at an MOI of 0.1. The data demonstrate that pACE-yCD(T5.0000)<pAC3-yCD1 (T5.0001)<pAC3-CD2 (T5.0002) in terms of thespecific activity of cytosine deaminase in tissue culture cells.

Example 6 Vector Purification and Concentration

A vector of the disclosure is manufactured by transient transfection on293T cells (Example 3), followed by harvesting of the cell supernatant,filtration, benzonase treatment, diafiltration/concentration anddialysis. A further chromatography column step may be included, known tothose skilled in the art (see for example U.S. Pat. No. 5,792,643; T.Rodriguez et al. J Gene Med 9:233 2007; WO2010148203. Vector is alsoproduced from a permanently infected cell line and processed as above(see for example WO2010148203). Clinical material is released based onstandard testing such as sterility, mycoplasma and endotoxins, plusproduct specific potency, strength, and identity testing. Titer isdetermined as Transducing Units (TU) by PCR quantitation of integratedviral vector DNA in target cells (Example 4). The final product istargeted to have a titer of up to 10⁹ TU/ml formulated in isotonicTris-buffered sucrose solution, as a sterile injectable.

In general, to accurately and precisely determine the strength of vectorlots, a quantitative PCR-based titer assay has been developed (describedin general terms in example 4). The details of the assay procedureconsist of the following steps:

Transduction.

Transductions are performed in a 12-well plate format using the stablehuman prostate adenocarcinoma derived PC-3 cell line. For each testsample, three dilutions of un-titered vector preparation are used totransduce PC-3 cells in triplicate wells. Viral replication is stopped24 hours post-transduction with azidothymidine (AZT). Cells aremaintained for an additional 24-64 hours prior to harvesting and genomicDNA purification.

Genomic DNA Preparation.

Qiagen DNeasy DNA Minikits are used to prepare genomic DNA fromtransduced harvested cells as per the manufacturer's protocol. DNAconcentrations and quality are assessed by direct absorbance measurementusing UV/vis spectrophotometry to determine the A260 and A260/A280ratio.

Real-Time Quantitative PCR.

The BioRad CFX96 real-time PCR instrument or equivalent is used forperforming quantitative PCR. Provector copy numbers present in each testsample are measured by using specific DNA oligonucleotide primers inconjunction with a TaqMan probe designed to amplify the integrated, orpro-retroviral, U3/Psi packaging versus the CMV/Psi plasmid promoter.Vector titer is expressed relative to a copy number standard curve. Togenerate the vector copy number standard curve, genomic DNA from PC-3cells is spiked with a unique plasmid containing the pro-retroviralU3/Psi sequence. Vector test sample titers are obtained by calculatingthe number of transduced genomes in multiple dilutions using multiplereactions per dilution.

For each titer assessment, a non template control (wells containing allcomponents except plasmid or genomic DNA) and a negative control (allcomponents including equivalent genomic DNA from non-transduced PC-3cells), is performed in triplicate. The titer values are expressed intransduction units per milliliter (TU/mL).

The potency of the vector of the disclosure is dependent on both thereplication of the vector and the resultant cytosine deaminase (CD)activity in target cells. Therefore the potency assay measures theincrease in CD activity over time as vector infection spreads in apreviously uninfected cell line in tissue culture. The assay measuresthe enzymatic activity of the transferred yCD2 protein in transducedcells during early, middle and late stages of infection by monitoringthe conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU),using reverse phase HPLC separation with UV detection. The increase ofCD activity over the course of the infection is a function of thepercent of cells infected over time and indicative of the TOCA 511vector's ability to replicate. CD activity based on the 5-FC to 5-FUconversion rate is measured for each time point in CD units per mg ofprotein (the specific activity, SA). The increase in SA is then plottedover time, and reflects both the increase in the percentage of cellstransduced as a result of viral replication in the culture, and theresultant transfer of CD activity. Accumulated data from multiple assaysand vector lots has been used to determine an appropriate specificationfor this increase in SA of CD, for product release. The assay has 1, 3and 5 day timepoints after an initial infection at an MOI of about 0.1and a non-infected control.

CD activity from late stage infected cells (day 5 time point) wascompared between lots to evaluate the use of this activity as anIdentity test. The assay includes the following steps:

Transductions.

Transductions are performed in multi-well plate format on U87 cells. Foreach transduction, three suitable dilutions are used and each performedin triplicate. Cells are harvested at 0, 1, 3 and 5 days posttransduction.

Set-Up of CD Reaction.

Cells are lysed and the total protein concentration determined using theBCA protein assay using BSA as a standard. For the yCD2 enzyme assay, anappropriate amount of cell lysate is added to buffer containing 5-FCsuch that the rate of 5-FU formation remains linear over 1-2 hours at37° C. The final volume for the reaction mixture is 100 μL. After 2 h,the enzyme reaction is terminated by the addition of trichloroaceticacid, briefly vortexed and prepared for subsequent HPLC analyses. Celllysates from non-transduced cells are used as a negative control while asimilar assay using samples from 100% infected cells is used as apositive control.

HPLC Analysis.

The terminated reaction mixture is centrifuged at 12,000 rpm for 5minutes at room temperature in a micro-centrifuge. The supernatant isthen decanted away from the pellet and passed through a 0.2p filter tofurther remove particulates before injection onto a reverse phase HPLCcolumn previously equilibrated with an aqueous based mobile phasecontaining phosphate buffer at a pH around 4.0. The chromatograms arefollowed at 260 nm and 280 nm to monitor both substrate consumption andproduct formation. Concentrations of either substrate or product aredetermined using the graphing and analysis capabilities of GraphPad bycomparing them to previously generated standard curves calculated fromknown substrate or product concentrations. Amounts of 5-FU generatedover 1-2 h are used to determine CD units of activity (1 unit of CDactivity is defined as the formation 1 nmol of 5-FU per min) and theSpecific Activity is calculated dividing this number by the amount ofprotein (from the cell lysate) used in the assay.

Example 7 Construction and Use of a Vector Encoding a Single ChainAntibody to CTLA-4 (CD 152)

Single chain antibodies are derived from known full antibody sequencesthat have a desired effect. Such sequences are available (e.g.WO2006048749, US2006165706, U.S. Pat. No. 7,034,121, Genbank AccessionNumbers DJ437648, CS441506, CS441500, CS441494, CS441488, thedisclosures of which are incorporated herein by reference). Suchconventional antibody gene sequences are converted into single chainantibody (scFv) sequences by commonly used methods known to thoseskilled in the art (see for example Gilliland et al. “Rapid and reliablecloning of antibody variable regions and generation of recombinantsingle chain antibody fragments.” Tissue Antigens 47, 1-20, 1996). Phagesingle chain antibodies to CTLA-4 are also available from screeningphage-scFv libraries directly (Pistillo et al. Tissue Antigens 55:2292000), and can be used directly for insertion into the replicatingretroviral vectors of the disclosure. Regardless of how the sequence isderived, scFv are typically about 700-900 bp in length and aresynthesized by a commercial vendor (BioBasic) with a PsiI site at the 5′end and compatible NotI site at the 3′ end, as described previously.This sequence is then inserted into the replicating retroviral back bonefrom pAC3-yCD2 at the PsiI-NotI sites after removal of the yCD2sequence. Vector is produced and titered as described, and furtherpurified if necessary as described above. Human and Mouse CTLA4 are veryhomologous in sequence and the replicating retrovirus of the disclosureis first tested in a suitable syngeneic immunocompetent mouse modelssuch as the CT26/BALB/c model and S91 mouse melanoma models, well knownto those skilled in the art (see for example Hodge et al J. Immunol.174:5994 2005). Outcome is measured by one or more of: modulation oftumor growth; lack of toxicity; generation of antitumor responses;shrinkage of remote lesions indicating systemic immunity. Doses are inthe range of 10³ to 10⁷ TU in mice. In patients the vector isadministered by intralesional injection into tumor, or by administrationinto the circulation that then carries the virus to the tumor. Doses arein the range of 10⁵ to 10¹¹ TU.

Example 8 Anti-Melanoma Efficacy Studies with Anti CD152 Single ChainAntibody Expressing Vector in a Mouse Melanoma Model

Objective.

The objective of this study is to assess the effect of a novel MLV basedreplication-competent retroviral vector carrying single chain antibodydirected against Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) also referredto as Cluster of differentiation 152 (CD152) sequence (pAC3-αCD152) onmelanoma growth, when delivered via intratumoral (IT) injection in DBA/2mice bearing subcutaneous melanoma (Cloudman S91).

Mice.

Female DBA/2 or BALB/c mice (age ˜8 weeks) are purchased from JacksonLaboratories (Bar Harbor, Me.). Mice are acclimated for 7 days afterarrival before start of studies.

Cells.

Cloudman S91 cells (ATCC, Manassas Va.) are a spontaneously arisingmelanoma derived from DBA/2 mice. Cells are cultured in Dulbecco'smodified Eagles medium with 10% fetal bovine serum, sodium pyruvate, andGlutamax (Hyclone, Logan Utah, and Invitrogen, San Diego Calif.). Cellsare resuspended in PBS (Hyclone, Logan Utah) for implantation. S91 cells(1E6 in 100 μL) are injected into the right flank of DBA/2 mice.

Vector.

Vectors preparations are made by transient transfection (or from aproducer cell line in HT1080 cells) with titers of approximately7E6TU/ml. For initial studies vector is not further purified orconcentrated. For follow on experiments to determine full dose responsecurves, high titer purified material is prepared with a titer expectedaround 10⁸/ml. Vector is administered IT in a volume of 50-100 μL and IVin 100 μL the total dose/mouse of approximately 7E5 to 7E6 to7ETU/mouse. Vector expressing αCD152 is identified as Toca αCD152.

Tumor Implantation and Vector Injection.

Five groups of female DBA/2 (55 mice, 9-10 weeks of age) are implantedsubcutaneously with S91 melanoma cells (Day 0) and then dosed (day 4-7depending on growth rate of the S91 tumor; approximately 50-100 mm³)with vehicle (Groups 1), with control vector [AC3-GFP(V), (Group2),intratumor (IT) Toca αCD152 vector injection (Groups 3), or intravenousToca αCD152 vector injection (group 4). Group 5 mice have no tumorimplanted and are intravenously injected with Toca αCD152 only.

Data Analysis.

Tumor growth analysis is carried out to 2000 mm³ or to 60 days based onwhich ever comes first. 10 mice from each group will be plotted fortumor size over time. Statistical significance will be determined usinganalysis of variance (ANOVA). P values of <0.05 are consideredstatistically significant in all analyses, which are performed withPrism 5 statistical software (GraphPad Software) or equivalent. In-lifeobservations are also taken to assess any adverse events to αCD152expression during treatment.

Results.

Delivery of αCD152 by replicating MLV IT shows a statisticallysignificant retardation of growth compared to the controls. Delivery ofαCD152 by replicating MLV intravenously shows a statisticallysignificant retardation of growth compared to the controls abrogatesmelanoma burden from the DBA/2—Cloudman S91 mouse melanoma model.Further animal studies were performed as described more fully below.

Example 9 AC3-yCD2 Viral Vector is Therapeutic in an Intracranial HumanXenograft (U87) in Nude Mice

An intracranial xenograft model using the U87 human glioma cell line wasestablished to test RCR vector spread and biodistribution as well astherapeutic efficacy of RCR-vector mediated cytosine deaminase suicidegene therapy in a nude mouse host.

Following acclimation, mice were randomly assigned to one of 8 Treatmentgroups (see group description below). Seven groups underwentintracranial administration into the right striatum of 1×10⁵ U87 cellsadministered/mouse on Day 0. Group 8 mice were not implanted with tumor.At Day 5, mice were injected with Formulation Buffer only, or an RCRvector at 9×10⁵/5 μl, 9×10⁴/5 μl, or 9×10³/5 μl. Mice receiving novector, or vector at 9×10⁵/5 μl or 9×10³/5 μl were randomized to receive5-FC (500 mg/kg/day), administered as a single IP injection, beginningon Day 19, or no 5-FC. Mice receiving vector at mid dose all received5-FC (i.e., No separate control group for this dose). 5-FCadministration continued daily for 7 consecutive days followed by 15days of no treatment. Cycles of drug plus rest were repeated up to 4cycles. 10 mice from each group except group 8 were randomly assigned tothe survival analysis category. The remaining mice were sacrificedaccording to a predetermined schedule.

Group Assignments and Dose Levels N per Analysis Category Test Drug(A)Survival (B)Scheduled Group article Volume TX N analysis Sacrifice 1Form 5 μl none 4 4 before buffer first drug cycle 2 Form 5 μl 5-FC 10 10buffer 3 T5.0002 9e5/5 μl PBS 10 10 4 T5.0002 9e5/5 μl 5FC 25 10 3before start of each cycle, 15 total 5 T5.0002 9e4/5 μl 5FC 10 10 6T5.0002 9e3/5 μl 5FC 25 10 3 before start of each cycle, 15 total 7T5.0002 9e3/5 μl PBS 10 10 8 NO none 5FC 15 3 before TUMOR start of eachcycle, 15 total Total Number of Animals 109 60 49

Intravenous dosing was done via injection into the tail vein.Intraperitoneal dosing was done via injection into the abdomen with caretaken to avoid the bladder. For intracranial injection mice wereanesthetized with isoflurane and positioned in a stereotaxic device withblunt ear bars. The skin was shaved and betadine was used to treat thescalp to prepare the surgical site. The animal was placed on a heatingpad and a scalpel used under sterile conditions to make a midlineincision through the skin. Retraction of the skin and reflection of thefascia at the incision site will allow for visualization of the skull. Aguide cannula with a 3 mm projection, fitted with a cap with a 3.5 mmprojection, will be inserted through a small burr hole in the skull andattached with dental cement and three small screws to the skull. Afterhardening of the cement, the skin will be closed with sutures. Theprojected stereotaxic coordinates are AP=0.5-1.0 mm, ML=1.8-2.0 mm,DV=3.0 mm. Exact stereotaxic coordinates for the cohort of animalsreceived will be determined in a pilot experiment (2-3 animals) byinjecting dye and determining its location. The animals will bemonitored during anesthesia recovery. Analgesics, buprenorphine, will beadministered subcutaneously (SC) before the end of the procedure thenbuprenorphine will be administered approximately every 12 hrs for up to3 days. Animals will be monitored on a daily basis. Cells or vector wereintracranially infused through an injection cannula with a 3.5 mmprojection inserted through the guide cannula. The rate was controlledwith a syringe pump fitted with a Hamilton syringe and flexible tubing.For cell injection, 1 microliter of cells was delivered at a flow rateof 0.2 microliters per minute (5 minutes total). For vector injection, 5microliters of vector was delivered at a flow rate 0f 0.33 microlitersper minute (15 minutes total).

Vector was delivered and calculated as transforming units (TU) per gramof brain weight to the mice. Using such calculation the translation ofdose can be calculated for other mammals including humans. FIG. 8 showsthe effect on vector dose in this mouse model.

Example 10 AC3-yCD2(V) is Therapeutic in a Syngeneic Mouse Model ofBrain Cancer

An intracranial implant model using the CT26 colorectal cancer cell linein syngeneic BALB/c mice was established to test RCR vector spread andbiodistribution as well as therapeutic efficacy of RCR-vector mediatedcytosine deaminase suicide gene therapy and its immunological impact.

This study included 129 animals, 0 Male, 119 Female and 10 contingencyanimals (10 Female). Following acclimation, mice were randomly assignedto one of 8 Treatment groups (see group description below). Seven groupsunderwent intracranial administration into the right striatum of 1×10⁴CT26 cells administered/mouse on Day 0. Group 8 mice were not implantedwith tumor. At Day 4, mice were injected with Formulation Buffer only,or vector at 9×10⁵/5 μl, 9×10⁴/5 μl, or 9×10³/5 μl. Mice receiving novector, or vector at 9×10⁵/5 μl or 9×10³/5 μl were randomized to receive5-FC (500 mg/kg/BID), administered by IP injection, beginning on Day 13,or no 5-FC. Mice receiving vector at mid dose received 5-FC (ie. Noseparate control group for this dose). 5-FC administration continueddaily for 7 consecutive days followed by 10 days of no treatment. Cyclesof drug plus rest were repeated up to 4 cycles. 10 mice from each groupexcept group 8 were randomly assigned to the survival analysis category.The remaining mice were sacrificed according to a predeterminedschedule.

Naïve sentinel mice were co-housed with the scheduled sacrifice animalsand taken down at the same time points to assess vector transmittalthrough shedding.

Group Assignments and Dose Levels N per Analysis Category Test Drug(A)Survival (B)Scheduled (C) Group article Volume TX N analysisSacrifice Sentinels 1 Form 5 μl PBS 4 4 before buffer first drug cycle 2Form 5 μl 5FC 10 10 buffer 3 T5.0002 9E5/5 μl PBS 10 10 4 T5.0002 9E5/5μl 5FC 25 10 3 before 1 before start of each start of each cycle, 15cycle, 5 total total 5 T5.0002 9E4/5 μl 5FC 10 10 6 T5.0002 9E3/5 μl 5FC25 10 3 before 1 before start of each start of each cycle, 15 cycle, 5total total 7 T5.0002 9E3/5 μl PBS 10 10 8 NO none 5FC 15 3 before TUMORstart of each cycle, 15 total Total Number of Animals 119 60 49 10

Intravenous dosing was done via injection into the tail vein.Intraperitoneal dosing was done via injection into the abdomen with caretaken to avoid the bladder. For intracranial administration, mice with aguide cannula with a 3.2 mm projection implanted into the rightstriatum, and fitted with a cap with a 3.7 mm projection were used. Theprojected stereotaxic coordinates are AP=0.5-1.0 mm, ML=1.8-2.0 mm,DV=3.2 mm (from bregma). Cells or vector were intracranially infusedthrough an injection cannula with a 3.7 mm projection inserted throughthe guide cannula. The rate was controlled with a syringe pump fittedwith a Hamilton syringe and flexible tubing.

For cell injection, 1 microliter of cells was delivered at a flow rateof 0.2 microliter per minute (5 minutes total). For vector injection, 5microliter of vector was delivered at a flow rate of 0.33 microliter perminute (15 minutes total).

Vector was delivered and calculated as transforming units (TU) per gramof brain weight to the mice. Using such calculation the translation ofdose can be calculated for other mammals including humans. FIG. 9 showsthe effect on vector dose in this mouse model when the vector isdelivered intracranially.

Example 11 Construction and Testing of RCR Vectors Expressing miR-128-1and miR128-2

Construction of Recombinant Replication Competent Retroviral VectorContaining a Heterologous Polynucleotide Sequence of Humanpri-miRNA-128-1.

The replication competent retroviral vector, pAC3-miR-128-1 expressingmiR-128-1 was derived from the backbone of pAC3-yCD2 described in one ofthe embodiments. The pAC3 backbone in the pAC3-miR-128-1 vector wasisolated by endonuclease digestion of the pAC3-yCD2 plasmid DNA with MluI and Not I to remove the IRES-yCD2 polynucleotide sequence. Thepolynucleotide DNA sequence of pri-miR-128-1 was obtained from theproduct sheet of the pEP-mir-128-1 expression vector (Cell BioLabs Inc.)(SEQ ID NO: 31). DNA sequence of pri-miR-128-1 was synthesized with aMlu I restriction enzyme site at the 5′ end and a Not I restrictionenzyme site at the 3′ end of the double-stranded DNA fragment forsubsequent insertion at the corresponding site in the Mlu I and Not Idigested pAC3-yCD2 plasmid DNA described above. The resulting construct,pAC3-miR-128-1, encodes 3 genes: the gag, the pol, and the env, and thenon-coding pri-miR-128-1 sequence.

Testing of Expression of Mature miR-128 from Cells Transduced withmiR-128 Containing Recombinant Replication Competent Retroviral Vector.

In order to confirm the expression of miR-128 from cells transduced withmiR-128 containing recombinant replication competent retroviral vectors,cells from day 9 post infection at which the maximal infectivity hasreached were expanded and harvested to extract total RNA for detectionof mature miRNA expression. The results from Taqman microRNA assayshowed an over expression of mature miR-128 from both HT1080 and U87-MGcells transduced with pAC3-miR-128-1, pAC3-miR-128-2, andpAC3-H1-shRNAmiR128 vectors, respectively, compared to untransducedcells. In both cell lines, cells transduced with pAC3-miR-128-1 andpAC3-H1-shRNAmiR128 vector expressed higher level of mature miR-128 thancells transduced with pAC3-miR-128-2 vector.

Example 12 Construction and Testing of Recombinant Replication CompetentRetroviral Vector Containing Heterologous Polynucleotide Sequences ofIRES, yCD2, Human H1 Promoter and Human Pre-miR128-1

Construction.

The replication competent retroviral vector, pAC3-yCD2-H1-shRNAmiR128 isderived from the backbone of pAC3-yCD2 described in one of theembodiments. The pAC3-yCD2 backbone in the pAC3-yCD2-H1-shRNAmiR128vector is isolated by endonuclease digestion of the pAC3-yCD2 plasmidDNA with Not I. The polynucleotide DNA sequence of the human H1 promoteris obtained from the product information of pSilencer 3.1 H1 hygroexpression vector (Ambion), and the polynucleotide DNA sequence of theshort hairpin structured pre-miR-128-1 is obtained from thehttp:(//)www.mirbase.org/. DNA sequence of pre-miR128-1 linked to thehuman H1 promoter (SEQ ID NO: 34) is synthesized with a Not Irestriction enzyme site at both ends of the double-stranded DNA fragmentfor subsequent insertion at the corresponding site in Not I digestedpAC3-yCD2 plasmid DNA described above. The resulting construct,pAC3-H1-shRNAmiR128, encodes 4 genes: the gag, the pol, and the env, andthe yCD2, and the non-coding short hairpin structured pre-miR-128-1sequence.

Vector stock is produced by transient transfection of thevector-encoding plasmid DNA into 293T cells using calcium phosphatemethod. Eighteen hours post transfection, the culture is replaced withfresh medium. Twenty-four hours post medium replacement, the supernatantcontaining the vector is collected and filtered through a 0.45 μm filterand used immediately or stored in aliquots at −80° C. for later use.Twenty micro-liter of the collected vector stocks is used to infecthuman prostate cancer cells, PC3. Twenty-four hours post infection, AZTis added to the cells to inhibit further viral replication. Forty-eighthours post infection, genomic DNA of infected PC3 cells is extracted fortiter assay. The titer of the vector stocks is determined by qPCR withan inclusion of standards of known copy numbers.

Testing of Replication Kinetics of the pAC3-yCD2-H1-shRNAmiR128Recombinant Replication Competent Retroviral Vectors in Culture.

In order to confirm that the incorporation of H1-pre-miR-128-1replicates normally, calculated volume of each vector stocks collectedfrom transient transfection mentioned above is used to infect freshhuman fibrosarcoma cells, HT1080 and human glioma cells, U87-MG,respectively, at a MOI of 0.1. Transduced cells are passaged at day 3, 6and 9 post infection. At each time point, a portion of cells arecollected for genomic DNA extraction for qPCR. Dilutions of genomic DNAare made to generate aliquots of genomic DNA with same concentration forequal amount of genomic in-put in qPCR. Replication kinetics of eachvectors are generated by plotting inversed C(t) values vs. time points.Result show that the vector replicates at similar kinetics compared tocontrol MLV virus.

Testing of Expression of Mature miR-128 from Cells Transduced with thepAC3-yCD2-H1-shRNAmiR128 Recombinant Replication Competent RetroviralVector.

To confirm the expression of miR-128 from cells transduced withpAC3-yCD2-H1-shRNAmiR128 recombinant replication competent retroviralvector, cells from day 9 post infection, at which the maximalinfectivity is reached, are expanded and harvested to extract total RNAfor detection of mature miRNA expression. Result from Taqman microRNAassay shows an over expression of mature miR-128 from both HT1080 andU87-MG cells transduced with the pAC3-yCD2-H1-shRNAmiR128compared tountransduced cells.

Testing of Bmi-1 Expression from Cells Transduced withpAC3-yCD2-H1-shRNAmiR128 Recombinant Replication Competent RetroviralVectors to Demonstrate Target Engagement of miR-128.

Bmi-1 expression has been observed to be up-regulated in a variety ofhuman cancers including glioblastoma, and has been shown to be thetarget of miR-128. To confirm target engagement of miR-128, Bmi-1expression from cells transduced with pAC3-yCD2-H1-shRNAmiR128 isdetected by qRT-PCR. The result shows that U87-MG cells transduced withpAC3-yCD2-H1-shRNAmiR128 express lower level of Bmi-1 than untransducedcells, whereas in HT1080 cells no significant difference was observedbetween transduced and untransduced cells. The data support the conceptthat miR-128 plays an important functional role in the central nervoussystem.

Testing of yCD2 Expression from Cells Transduced withpAC3-yCD2-H1-shRNAmiR128 by Immune-Blot.

To confirm the expression of yCD2 from cells transduced withpAC3-yCD2-H1-shRNAmiR128 recombinant replication competent retroviralvector, cells from day 9 post infection, at which the maximalinfectivity is reached, are expanded and harvested to extract totalprotein for detection of yCD2 expression. The result from immune-blotshows normal expression yCD2 from both HT1080 and U87-MG cellstransduced with the pAC3-yCD2-H1-shRNAmiR128 compared to pAC3-yCD2transduced cells.

Example 13 Anti-Tumor Efficacy Studies with miRNA Expressing Vector in aMouse/Human Xenograft Model

Objective.

The objective of this study is to assess the effect of a novel MLV basedreplication-competent retroviral vectors carrying the miR128 sequence(AC3-miR128-1(V); AC3-miR128-2(V); AC3-miR128-3(V) on survival, whendelivered via intracranial (IC) injection in nude mice bearing a humanglioma xenograft, at three Toca 511 dose levels.

Mice.

Female athymic nude-Foxn1̂nu (nude) mice (age ˜8 weeks) are purchasedfrom Harlan (Indianapolis Ind.). Mice are acclimated for 7 days afterarrival. Mice undergo surgical placement of an indwelling guide cannulawith a 3.0 mm projection implanted into the right striatum, and fittedwith a cap containing a 3.5 mm projection. The stereotaxic coordinatesare AP=+0.5 mm, ML=−1.8 mm (from bregma).

Cells.

U-87 MG cells (ATCC, Manassas Va.) are derived from a malignant gliomafrom a 44 year old Caucasian female. Cells are cultured in Dulbecco'smodified Eagles medium with 10% fetal bovine serum, sodium pyruvate, andGlutamax (Hyclone, Logan Utah, and Invitrogen, San Diego Calif.). Cellsare resuspended in PBS (Hyclone, Logan Utah) for implantation. U-87 MGcells (1E5 in 1 μL) are infused at 0.2 μL per minute (5 minutes,followed by a hold of 5 minutes) IC through an injection cannula with a3.5 mm projection inserted through the guide cannula.

Vectors preparations are made by transient transfection (or from aproducer cell line) and all have titers of approximately 5E6TU/ml. Forinitial studies vector is not further purified or concentrated. Forfollow on experiments to determine full dose response curves, high titerpurified material is prepared with a titer of around 10E8/ml. Vector isadministered IC in a volume of 5 ul or less for a minimum totaldose/mouse of approximately 2.5E4 TU/mouse.

Tumor Implantation and Vector Injection.

Six groups of female athymic nude-Foxn1̂nu mice (65 mice, 9-10 weeks ofage) are implanted IC with U-87 tumor cells (Day 0) then dosed IC (day4-7 depending on growth rate of the U87 cells) with vehicle (Groups 1),with control vector (AC3-GFP(V), Group2) or IC with AC3-miR128-1(V);AC3-miR128-2(V); AC3-miR128-3(V) (Groups 3-5). Group 6 mice were notimplanted with tumor or vector.

Data Analysis.

Survival analysis to day 60 is performed on 10 mice each from Groups 1-6and plotted as a Kaplan Meyer plot. Survival curves are compared by thelog-rank test. P values of <0.05 are considered statisticallysignificant in all analyses, which are performed with Prism 5statistical software (GraphPad Software) or equivalent.

Results from treatment with the vectors show a statistically significantsurvival advantage in this human glioma xenograft model compared totreatment with control vector or vehicle alone.

Example 14 Thymosin Combination Therapy

Experiments using Thymosin Alpha 1 were performed in conjunction withToca 511 treatment in the Tu2449/B6C3F1 glioma mouse model (U. Pohle etal. Int J Oncol. 15:829-834 (1999); HM. Smilowitz et al. J. Neurosurg106:652-659 2007). Experiments were conducted in a similar manner tothose in the BALB/c-CT26 model (Example 10), except that the initialintracranial cell innoculum was at 10⁴ cells and the 5-FC dosing wastwice a day (BID) intra-peritoneally at 500 mg/kg, with 10 days off drugfollowed by 4 days with 5-FC administration. In addition to theadministration of vector and 5-FC some groups were dosed with thymosinalpha 1(TA1). Thymosin Alpha 1 was obtained from Sigma Aldrich cat#T3641 and a stock solution made in sterile water at 400 μg/mL. TA1 (200μg/kg, ˜40 μg/animal) was given IP starting on day 7 SID for 28 days.

The results are presented in FIG. 10. Using a suboptimal dose of Toca511(E3) and standard BID 5-FC dosing, the addition of thymosin alpha 1increased the survival rate to that of the optimal E5 dosing of Toca511. When compared to buffer only animals there was a significantsurvival advantage (p=0.0016, hazard ratio 0.1111, 95% CL 0.028 to0.436). Further, using a suboptimal dose of Toca511 (E3), thymosin alpha1 dosing, and standard BID 5-FC dosing resulted in a survival advantagecompared to a suboptimal dose of Toca511 (E3) and only thymosin alpha 1dosing.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the disclosure.Accordingly, other embodiments are within the scope of the followingclaims.

1. A therapeutic combination comprising a thymosin-1-alpha polypeptideand a replication retroviral vector for use in the treatment of asubject comprising a cell proliferative disease or disorder, wherein thereplication competent retroviral vector comprises a retroviral GAGprotein; a retroviral POL protein; a retroviral envelope; a retroviralpolynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′end of the retroviral polynucleotide sequence, a promoter sequence atthe 5′ end of the retroviral polynucleotide, said promoter beingsuitable for expression in a mammalian cell, a gag nucleic acid domain,a pol nucleic acid domain and an env nucleic acid domain; a cassettecomprising an internal ribosome entry site (IRES) operably linked to aheterologous polynucleotide, wherein the cassette is positioned 5′ tothe 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviralenvelope; and cis-acting sequences necessary for reverse transcription,packaging and integration in a target cell.
 2. The combination of claim1, wherein the heterologous polynucleotide comprises a suicide gene thatexpresses a polypeptide that converts a non-toxic prodrug to a toxicdrug.
 3. The combination of claim 1, wherein the target cell is a cancercell.
 4. The combination of claim 1, wherein the target cell comprises acell proliferative disorder.
 5. The combination of claim 4, wherein thecell proliferative disorder is selected from the group consisting oflung cancer, colon-rectum cancer, breast cancer, prostate cancer,urinary tract cancer, uterine cancer, brain cancer, head and neckcancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer,rheumatoid arthritis or other autoimmune disease.
 6. The combination ofclaim 1, wherein the retroviral vector is administered prior to thethymosin-alpha-1 polypeptide.
 7. The combination of claim 1, wherein theretroviral polynucleotide sequence is derived from murine leukemia virus(MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus(FeLV) Baboon endogenous retrovirus (BEV), porcine endogenous virus(PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus,Xenotropic murine leukemia virus-related virus (XMRV), avianreticuloendotheliosis virus (REV), or Gibbon ape leukemia virus (GALV).8. The combination of claim 1, wherein the retroviral envelope is anamphotropic MLV envelope.
 9. The combination of claim 1, wherein theretrovirus is a gammaretrovirus.
 10. The combination of claim 1, whereinthe thymosin-alpha-1 polypeptide comprises at least 85% identity to SEQID NO:73 and having thymosin-alpha-1 activity.
 11. The combination ofclaim 1, wherein the heterologous polynucleotide encodes a polypeptidehaving cytosine deaminase activity.
 12. The combination of claim 1,wherein the heterologous polynucleotide is selected from the groupconsisting of a suicide gene and an immunopotentiating gene.
 13. Thecombination of claim 1, wherein the retrovirus further comprises anmiRNA.
 14. The combination of claim 1, wherein the replication competentretrovirus comprises a retroviral GAG protein; a retroviral POL protein;a retroviral envelope; a retroviral polynucleotide comprisingLong-Terminal Repeat (LTR) sequences at the 3′ end of the retroviralpolynucleotide sequence, a promoter sequence at the 5′ end of theretroviral polynucleotide, said promoter being suitable for expressionin a mammalian cell, a gag nucleic acid domain, a pol nucleic aciddomain and an env nucleic acid domain; a cassette comprising an internalribosome entry site (IRES) operably linked to a polynucleotide encodingcytosine deaminase, wherein the cassette is positioned 5′ to the 3′ LTRand 3′ to the env nucleic acid domain encoding the retroviral envelope;and cis-acting sequences necessary for reverse transcription, packagingand integration in a target cell.
 15. The combination of claim 1,wherein the thymosin-1-alpha and retroviral vector are formulated fordelivery simultaneously.
 16. A method of treating a subject with a cellproliferative disorder comprising: administering a thymosin-alpha-1polypeptide to the subject either before, during or after administrationof a replication competent retrovirus, the replication competentretrovirus comprising a retroviral GAG protein; a retroviral POLprotein; a retroviral envelope; a retroviral polynucleotide comprisingLong-Terminal Repeat (LTR) sequences at the 3′ end of the retroviralpolynucleotide sequence, a promoter sequence at the 5′ end of theretroviral polynucleotide, said promoter being suitable for expressionin a mammalian cell, a gag nucleic acid domain, a pol nucleic aciddomain and an env nucleic acid domain; a cassette comprising an internalribosome entry site (IRES) operably linked to a heterologouspolynucleotide, wherein the cassette is positioned 5′ to the 3′ LTR and3′ to the env nucleic acid domain encoding the retroviral envelope; andcis-acting sequences necessary for reverse transcription, packaging andintegration in a target cell.
 17. The method of claim 16, wherein theretroviral polynucleotide sequence is derived from murine leukemia virus(MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus(FeLV) Baboon endogenous retrovirus (BEV), porcine endogenous virus(PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus,Xenotropic murine leukemia virus-related virus (XMRV), avianreticuloendotheliosis virus (REV), or Gibbon ape leukemia virus (GALV).18. The method of claim 16, wherein the retroviral envelope is anamphotropic MLV envelope.
 19. The method of claim 16, wherein theretrovirus is a gammaretrovirus.
 20. The method of claim 16, wherein thetarget cell is a neoplastic cell.
 21. The method of claim 16, whereinthe cell proliferative disorder is selected from the group consisting oflung cancer, colon-rectum cancer, breast cancer, prostate cancer,urinary tract cancer, uterine cancer, brain cancer, head and neckcancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer,rheumatoid arthritis or other autoimmune disease.
 22. The method ofclaim 16, wherein the thymosin-alpha-1 polypeptide comprises at least85% identity to SEQ ID NO:73 and having a thymosin-alpha-1 activity. 23.The method of claim 16, wherein the heterologous polynucleotide encodesa polypeptide having cytosine deaminase activity.
 24. The method ofclaim 16, wherein the heterologous polynucleotide is selected from thegroup consisting of a suicide gene and an immunopotentiating gene. 25.The method of claim 16, wherein the retrovirus further comprises anmiRNA.
 26. The method of claim 16, wherein the replication competentretrovirus comprising a retroviral GAG protein; a retroviral POLprotein; a retroviral envelope; a retroviral polynucleotide comprisingLong-Terminal Repeat (LTR) sequences at the 3′ end of the retroviralpolynucleotide sequence, a promoter sequence at the 5′ end of theretroviral polynucleotide, said promoter being suitable for expressionin a mammalian cell, a gag nucleic acid domain, a pol nucleic aciddomain and an env nucleic acid domain; a cassette comprising an internalribosome entry site (IRES) operably linked to a polynucleotide encodingcytosine deaminase, wherein the cassette is positioned 5′ to the 3′ LTRand 3′ to the env nucleic acid domain encoding the retroviral envelope;and cis-acting sequences necessary for reverse transcription, packagingand integration in a target cell.